Method for inhibiting bone resorption

ABSTRACT

The invention is directed to a method of inhibiting bone resorption. The method comprises administering to a human an amount of sclerostin inhibitor that reduces a bone resorption marker level for at least 2 weeks. The invention also provides a method of monitoring anti-sclerostin therapy comprising measuring one or more bone resorption marker levels, administering a sclerostin binding agent, then measuring the bone resorption marker levels. Also provided is a method of increasing bone mineral density; a method of ameliorating the effects of an osteoclast-related disorder; a method of treating a bone-related disorder by maintaining bone density; and a method of treating a bone-related disorder in a human suffering from or at risk of hypocalcemia or hypercalcemia, a human in which treatment with a parathyroid hormone or analog thereof is contraindicated, or a human in which treatment with a bisphosphonate is contraindicated.

CROSS-REFERENCE TO RELATED APPLICATIONS AND INCORPORATION BY REFERENCE

This application claims priority to U.S. Provisional Patent ApplicationNo. 60/973,024, filed Sep. 17, 2007.

The following applications are hereby incorporated by reference in theirentirety: U.S. patent application Ser. No. 11/410,540, filed Apr. 25,2006, which claims priority to U.S. Provisional Patent Application No.60/792,645, filed Apr. 17, 2006, U.S. Provisional Patent Application No.60/782,244, filed Mar. 13, 2006, U.S. Provisional Patent Application No.60/776,847, filed Feb. 24, 2006, and U.S. Provisional Patent ApplicationNo. 60/677,583, filed May 3, 2005; and U.S. patent application Ser. No.11/411,003, filed Apr. 25, 2006, which claims priority to U.S.Provisional Patent Application No. 60/792,645, filed Apr. 17, 2006, U.S.Provisional Patent Application No. 60/782,244, filed Mar. 13, 2006, andU.S. Provisional Patent Application No. 60/776,847, filed Feb. 24, 2006.

TECHNICAL FIELD OF THE INVENTION

The invention generally relates to methods of using sclerostin bindingagents to modulate bone density.

BACKGROUND OF THE INVENTION

Loss of bone mineral content can be caused by a wide variety ofconditions and may result in significant medical problems. For example,osteoporosis is a debilitating disease in humans and is characterized bymarked decreases in skeletal bone mass and mineral density, structuraldeterioration of bone, including degradation of bone microarchitectureand corresponding increases in bone fragility (i.e., decreases in bonestrength), and susceptibility to fracture in afflicted individuals.Osteoporosis in humans is generally preceded by clinical osteopenia, acondition found in approximately 25 million people in the United States.Another 7-8 million patients in the United States have been diagnosedwith clinical osteoporosis. The frequency of osteoporosis in the humanpopulation increases with age. Among Caucasians, osteoporosis ispredominant in women who, in the United States, comprise 80% of theosteoporosis patient pool. The increased fragility and susceptibility tofracture of skeletal bone in the aged is aggravated by the greater riskof accidental falls in this population. Fractured hips, wrists, andvertebrae are among the most common injuries associated withosteoporosis. Hip fractures in particular are extremely uncomfortableand expensive for the patient, and for women, correlate with high ratesof mortality and morbidity.

SUMMARY OF THE INVENTION

The invention is directed to methods of using a sclerostin inhibitor forinhibiting bone resorption in humans. The method comprises administeringto a human an amount of sclerostin inhibitor that is effective to reducethe level of a marker of bone resorption and optionally increase thelevel of a marker of bone formation. In some embodiments, boneresorption is inhibited and bone formation is increased for at leastabout 7 days, 2 weeks, 3 weeks, 4 weeks, 1 month, 5 weeks, 6 weeks, 7weeks, 8 weeks, 2 months, 3 months or longer. In related embodiments,the invention provides a method of increasing bone mineral density ortreating a bone-related disorder. The invention further provides amethod of ameliorating the effects of an osteoclast-related disorder.The method comprises administering to a human a sclerostin inhibitorthat reduces the level of a marker of bone resorption compared to bonemarker levels absent treatment. The sclerostin inhibitor also increasesthe level of a marker of bone formation by at least about 10% comparedto bone marker levels absent treatment. The sclerostin inhibitor can beadministered via a single dose or in multiple doses. For example, thesclerostin inhibitor can be administered in a short-term therapy regimento, e.g., increase bone formation, and/or can be administered long-termto prevent loss of bone mineral density in a maintenance therapeuticregimen.

In any of the methods disclosed herein, the level of one or more markersof bone resorption is reduced by at least about 5%, 10%, 15%, 20%, 30%,40%, 50% or more for at least 2 weeks, 3 weeks, 30 days, 1 month, 6weeks, 2 months or longer, compared to pre-treatment levels or normallevels for that patient population. By way of non-limiting example, thelevel of the marker of bone resorption by 3 weeks after treatment isdecreased by, e.g., at least about 20% compared to pre-treatment levelsor normal levels for that patient population. In any of the precedingmethods, the level of the marker of bone formation is increased by atleast about 10%, about 20%, about 30%, about 40%, about 50%, about 60%,about 70%, about 80%, about 90%, about 100% or more for at least about 2weeks, 3 weeks, 30 days, 1 month, 6 weeks, 2 months or longer, comparedto pre-treatment levels or normal levels for that patient population. Byway of non-limiting example, the level of the marker of bone formationby 3 weeks after treatment is increased by, e.g., at least about 20%compared to pre-treatment levels or normal levels for that patientpopulation. In one exemplary embodiment, the marker of bone resorptionis serum level of C-telopeptide of type I collagen (CTX). In otherexemplary embodiments, the marker of bone formation is bone-specificalkaline phosphatase (BSAP), osteocalcin (OstCa), and/or N-terminalextension of procollagen type 1 (P1NP).

The invention also provides a method of treating a bone-relateddisorder, wherein the method comprises administering to a human one ormore amounts of a sclerostin inhibitor effective to increase bonemineral density for the total body (e.g., head, trunk, arms, and legs)or at the hip (e.g., total hip and/or femoral neck), spine (e.g., lumbarspine), wrist, finger, shin bone and/or heel by about 1%, about 2%,about 3%, about 4%, about 5%, about 6%, about 8%, about 10%, about 12%,about 15%, about 18%, about 20%, about 25%, or 30% or more. In someembodiments, the bone mineral density of the human before treatment ischaracteristic of osteoporosis or osteopenia, and one or more doses ofsclerostin inhibitor are administered in an amount and for a timeeffective to improve bone mineral density such that the bone mineraldensity is no longer characteristic of osteoporosis and/or osteopenia.For example, one or more doses may be administered for an initial timeperiod to increase bone mineral density to within 2.5, or one, standarddeviations of the density normal for a young adult (i.e., a T-score≧−2.5 or a T-score ≧−1, as defined below). In exemplary embodiments, theinitial time period is about 3 months or less, 6 months or less, 9months or less, 1 year or less, 18 months or less, or longer. The methodmay further comprise subsequently administering one or more amounts of asclerostin inhibitor effective to maintain bone mineral density,optionally for a maintenance time period of at least about 6 months, 1year, 2 years or longer (e.g., over the life-time of the subject).

The invention further provides a method of treating a bone-relateddisorder in a human by administering one or more doses between about 0.1to about 20 mg/kg, or about 0.1 to about 12 mg/kg, or about 0.5 to about12 mg/kg, or about 1 to about 10 mg/kg, or about 1 to about 8 mg/kg, orabout 2 to about 8 mg/kg, or about 3 to about 8 mg/kg. In someembodiments, doses may be administered at an interval of about once 2weeks or longer, once every month or longer, or once every 2 months orlonger, or once every 3 months or longer, or once every 4 months orlonger, or once every 5 months or longer, or once every 6 months orlonger, or once every 9 months or longer, or once every year or longer.The sclerostin inhibitor may be used in the preparation of a medicamentfor administration using any of the dosing and timing regimens describedherein. Optionally, the sclerostin inhibitor is presented in acontainer, such as a single dose or multidose vial, containing a dose ofsclerostin inhibitor for administration (e.g., about 70 to about 450 mgof sclerostin inhibitor). In one exemplary embodiment, a vial maycontain about 70 mg or 75 mg of sclerostin inhibitor, e.g.anti-sclerostin antibody, and would be suitable for administering asingle dose of about 1 mg/kg. In other embodiments, a vial may containabout 140 mg or 150 mg; or about 210 mg or 220 mg or 250 mg; or about280 mg or 290 mg or 300 mg; or about 350 mg or 360 mg; or about 420 mgor 430 mg or 440 mg or 450 mg of sclerostin inhibitor, e.g.,anti-sclerostin antibody.

Additionally, the invention provides a method of treating a bone-relateddisorder in a human suffering from or at risk of hypocalcemia orhypercalcemia, a human in which treatment with a parathyroid hormone oranalog thereof is contraindicated, or a human in which treatment with abisphosphonate is contraindicated. The method comprises administering tothe human an amount of a sclerostin inhibitor effective to increase thelevel of a marker of bone formation and/or reduce the level of a markerof bone resorption, without resulting in hypocalcemia or hypercalcemia(e.g., clinically-significant hypocalcemia or hypercalcemia).

The invention also provides a method of monitoring anti-sclerostintherapy, i.e., the physiological response to a sclerostin inhibitor. Themethod comprises the steps of administering one or more doses of asclerostin inhibitor, and detecting the level of one or more markers ofbone resorption, wherein a reduction of at least about 5%, about 10%,about 15%, about 20%, about 30%, about 40%, about 50% or more in thelevel of a marker of bone resorption, compared to pre-treatment levelsor normal levels for that patient population, is indicative of effectivetreatment. The method optionally further comprises the step of detectingthe level of one or more markers of bone formation, wherein an increaseof at least about 10%, about 20%, about 30%, about 40%, about 50%, about60%, about 70%, about 80%, about 90%, or about 100% in the level of amarker of bone formation, compared to pre-treatment levels or normallevels for that patient population, is indicative of effectivetreatment. In certain embodiments, the increase in bone formation markerlevels is about 20%. The method may further comprise the step ofadjusting the dose of a sclerostin inhibitor to a different amount,e.g., higher if the change in bone resorption and/or bone formation isless than desired, or lower if the change in bone resorption and/or boneformation is more than desired.

In a different aspect, the invention provides selected sclerostininhibitors that reduce the level of a marker of bone resorption by atleast about 5%, about 10%, about 15%, about 20%, about 30%, about 40%,about 50% or more and increase the level of a marker of bone formationby at least about 10%, about 20%, about 30%, about 40%, about 50%, about60%, about 70%, about 80%, about 90%, about 100%, or more, for at leastabout 1 week, about 2 weeks, about 1 month, about 6 weeks, about 2months, about 10 weeks, or about 3 months. In a related aspect, theinvention provides a method of selecting such sclerostin inhibitors byadministering a candidate sclerostin inhibitor to an animal andselecting a candidate sclerostin inhibitor that changes the level of amarker of bone resorption and/or formation to the desired extent.

In any of the preceding methods or embodiments of the invention, thesclerostin inhibitor may be a sclerostin binding agent. The use ofsclerostin binding agents disclosed in U.S. Patent Publication No.20070110747, e.g., in any of the methods disclosed herein or forpreparation of medicaments for administration according to any of themethods disclosed herein, is specifically contemplated. In this regard,the invention includes use of a sclerostin binding agent in preparationof a medicament for inhibiting bone resorption in an amount from about 1mg/kg to about 10 mg/kg, wherein the amount is effective to reduce serumlevel of C-telopeptide of type I collagen (CTX) by at least 20%,compared to pre-treatment or normal levels, by 3 weeks after treatmentbegins. The invention also includes use of a sclerostin binding agent inpreparation of a medicament for increasing bone mineral density in anamount from about 1 mg/kg to about 10 mg/kg, wherein the amount iseffective to (a) reduce serum level of CTX by at least 20% compared topre-treatment or normal levels, by 3 weeks after treatment begins, and(b) increase serum level of a bone formation marker selected from thegroup consisting of serum level of bone-specific alkaline phosphatase(BSAP), serum level of amino-terminal extension of peptide ofprocollagen type 1 (PINP), and serum level of osteocalcin (OstCa), by atleast 20%, compared to pre-treatment or normal levels, by 3 weeks aftertreatment begins.

The invention further includes use of a sclerostin binding agent inpreparation of a medicament for treating a bone-related disorder in anamount from about 1 mg/kg to about 10 mg/kg for a first period of time,wherein the amount is effective to increase bone mineral density at thehip, spine, wrist, finger, shin bone and/or heel by at least about 3%,followed by an amount of from about 1 mg/kg to about 10 mg/kg for asecond period of time effective to maintain bone mineral density. Use ofa sclerostin binding agent in preparation of a medicament for treating abone-related disorder in a human suffering from or at risk ofhypocalcemia or hypercalcemia in an amount from about 1 mg/kg to about10 mg/kg, also is contemplated, as well as use of a sclerostin bindingagent in preparation of a medicament for treating a bone-relateddisorder in (a) a human in which treatment with a parathyroid hormone oranalog thereof is contraindicated or (b) a human in which treatment withbisphosphonate is contraindicated.

The invention also includes containers comprising anti-sclerostinantibody or fragment thereof. In one embodiment, the container comprisesanti-sclerostin antibody or fragment thereof and instructions foradministering the antibody or fragment thereof in an amount effective to(a) reduce serum level of C-telopeptide of type I collagen (CTX) by atleast 20%, compared to pre-treatment or normal levels, by 3 weeks aftertreatment begins, and (b) increase serum level bone-specific alkalinephosphatase (BSAP), serum level of amino-terminal extension of peptideof procollagen type 1 (PINP), or serum level of osteocalcin (OstCa) byat least 20%, compared to pre-treatment or normal levels, by 3 weeksafter treatment begins. Alternatively or in addition, the containercomprises an amount of anti-sclerostin antibody from about 70 mg toabout 450 mg. The invention further provides a container comprisinganti-sclerostin antibody or fragment thereof and instructions foradministering the antibody or fragment thereof for treating abone-related disorder in an amount from about 1 mg/kg to about 10 mg/kgevery two or four weeks. In addition, the invention provides a containercomprising anti-sclerostin antibody or fragment thereof and instructionsfor administering the antibody or fragment thereof for treating abone-related disorder in an amount from about 1 mg/kg to about 10 mg/kgfor a period of about 3 months.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graph of percent change of N-terminal extension ofprocollagen type 1 (P1NP) levels compared to baseline and placebo P1NPlevels versus time (day) post-administration of various single doses ofa sclerostin binding agent in healthy, postmenopausal women.

FIG. 2 is a graph of percent change of bone-specific alkalinephosphatase (BSAP) levels compared to baseline and placebo BSAP levelsversus time (day) post-administration of various single doses of asclerostin binding agent in healthy, postmenopausal women.

FIG. 3 is a graph of percent change of osteocalcin levels compared tobaseline and placebo osteocalcin levels versus time (day)post-administration of various single doses of a sclerostin bindingagent in healthy, postmenopausal women.

FIG. 4 is a graph of percent change of serum C-terminal telopeptide oftype 1 collagen (CTX) levels compared to baseline and placebo serum CTXlevels versus time (day) post-administration of various single doses ofa sclerostin binding agent in healthy, postmenopausal women.

FIG. 5 are graphs of percent change of osteocalcin, BSAP, P1NP, and CTXlevels compared to baseline and placebo levels versus time (day)post-administration of a single dose of 5 mg/kg or 10 mg/kg ofsclerostin binding agent in healthy, postmenopausal women.

FIG. 6 is a graph of percent change of serum calcium levels compared tobaseline and placebo serum calcium levels versus time (day)post-administration of various single doses of a sclerostin bindingagent in healthy, postmenopausal women.

FIG. 7 are graphs of percent change of bone mineral density compared tobaseline and placebo versus time (day) post-administration of varioussingle doses of sclerostin binding agent in healthy, postmenopausalwomen.

DETAILED DESCRIPTION OF THE INVENTION

The invention is predicated, at least in part, on the surprisingdiscovery that blocking or inhibiting the biological activity of humansclerostin triggers multiple physiological responses linked to increasedbone mineral density (BMD), including significant inhibition of boneresorption. Most currently available therapies only inhibit boneresorption without increasing bone formation. Some currently availabletherapies for disorders associated with reduced BMD only increase boneformation without significantly reducing bone resorption. For example,when bone formation is triggered by some current drugs, bone resorptionmay also increase (albeit potentially at a lower rate than beforetherapy). In contrast, agents that interfere with sclerostin activityboth enhance bone formation and reduce bone resorption. In other words,sclerostin inhibitors “uncouple” bone formation and bone resorption tomore effectively build bone. The materials and methods of the inventionare superior to existing therapies whose therapeutic efficacy is limitedand which are accompanied by potentially serious adverse side effects.

In this regard, the invention provides a method of inhibiting boneresorption, e.g., bone resorption mediated by osteoclasts, bone cellsthat dissolve bone mineral matrices. The invention further provides amethod of ameliorating the effects of an osteoclast-related disorder,i.e., a disorder caused by abnormally increased osteoclast activitythat, in some embodiments, manifests as abnormally high bone resorption.The inventive method comprises administering to a human an amount ofsclerostin binding agent that reduces the level of a marker of boneresorption and, optionally, increases the level of a marker of boneformation.

Activity of a sclerostin inhibitor, e.g., a sclerostin binding agent,(further described below) may be measured in a variety of ways.Sclerostin binding agent-mediated increases in bone mineral content orbone density may be measured using single- and dual-energy X-rayabsorptometry, ultrasound, computed tomography, radiography, andmagnetic resonance imaging. The amount of bone mass may also becalculated from body weights or by using other methods (seeGuinness-Hey, Metab. Bone Dis. Relat. Res., 5:177-181 (1984)). Animalsand particular animal models are used in the art for testing the effectof the pharmaceutical compositions and methods on, for example,parameters of bone loss, bone resorption, bone formation, bone strength,or bone mineralization that mimic conditions of human disease such asosteoporosis and osteopenia. Examples of such models include theovariectomized rat model (Kalu, Bone and Mineral, 15:175-192 (1991);Frost and Jee, Bone and Mineral, 18:227-236 (1992); and Jee and Yao, J.Musculoskel. Neuron. Interact., 1:193-207 (2001)). The methods formeasuring sclerostin binding agent activity described herein also may beused to determine the efficacy of other sclerostin inhibitors.

In humans, bone mineral density can be determined clinically using dualx-ray absorptiometry (DXA) of, for example, the hip and spine. Othertechniques include quantitative computed tomography (QCT),ultrasonography, single-energy x-ray absorptiometry (SXA), andradiographic absorptiometry. Common central skeletal sites formeasurement include the spine and hip; peripheral sites include theforearm, finger, wrist and heel. Except for ultrasonography, theAmerican Medical Association notes that BMD techniques typically involvethe use of x-rays and are based on the principle that attenuation of theradiation depends on thickness and composition of the tissues in theradiation path. All techniques involve the comparison of results to anormative database.

Alternatively, a physiological response to one or more sclerostinbinding agents can be gauged by monitoring bone marker levels. Bonemarkers are products created during the bone remodeling process and arereleased by bone, osteoblasts, and/or osteoclasts. Fluctuations in boneresorption and/or bone formation “marker” levels imply changes in boneremodeling/modeling. The International Osteoporosis Foundation (IOF)recommends using bone markers to monitor bone density therapies (see,e.g., Delmas et al., Osteoporos Int., Suppl. 6:S2-17 (2000),incorporated herein by reference). Markers indicative of bone resorption(or osteoclast activity) include, for example, C-telopeptide (e.g.,C-terminal telopeptide of type 1 collagen (CTX) or serum cross-linkedC-telopeptide), N-telopeptide (N-terminal telopeptide of type 1 collagen(NTX)), deoxypyridinoline (DPD), pyridinoline, urinary hydroxyproline,galactosyl hydroxylysine, and tartrate-resistant acid phosphatase (e.g.,serum tartrate-resistant acid phosphatase isoform 5b). Boneformation/mineralization markers include, but are not limited to,bone-specific alkaline phosphatase (BSAP), peptides released from N- andC-terminal extension of type I procollagen (P1NP, PICP), and osteocalcin(OstCa). Several kits are commercially-available to detect and quantifymarkers in clinical samples, such as urine and blood.

Upon administration, the sclerostin binding agent preferably reduces thelevel of one or more markers of bone resorption, such as the serum levelof C-telopeptide of type I collagen (CTX). Accordingly, the inventionfurther provides a method of monitoring anti-sclerostin therapy, i.e.,the physiological response to a sclerostin binding agent or othersclerostin inhibitor. The method comprises administering a sclerostinbinding agent, then measuring the level of one or more markers of boneresorption. In addition, the method can comprise measuring the level ofone or more markers of bone formation before administration of asclerostin binding agent. The level of bone resorption marker duringand/or after treatment with the sclerostin binding agent may be comparedto a pre-treatment level, or alternatively may be compared to a standardrange typical of that patient population. One of ordinary skill in theart can readily determine a suitable standard range by testing arepresentative number of patients of like age, gender, disease level,and/or other characteristics of the patient population. The level ofbone resorption marker can be reduced by at least about 5% (e.g., about10%, about 20%, or about 30%) by a single dose of sclerostin bindingagent. In some embodiments, the dose of sclerostin binding agent reducesthe level of bone resorption marker at least about 40% (e.g., about 50%,about 60%, or about 70%) compared to the level of the bone resorptionmarker prior to administering the sclerostin binding agent. In addition,the bone resorption marker level may be reduced for at least about 3days (e.g., about 7 days, about 2 weeks, about 3 weeks, about 1 month,about 5 weeks, about 6 weeks, about 7 weeks, about 2 months, about 9weeks, about 10 weeks, about 11 weeks, or about 3 months) afteradministration of a single dose of the sclerostin binding agent.

In addition to decreasing the level of bone resorption markers, theamount of sclerostin binding agent administered to a patient also canincrease the level of one or more markers of bone formation, such as theserum level of BSAP, the serum level of P1NP, and/or the serum level ofOstCa. A single dose of sclerostin binding agent can increase the levelof a bone formation marker by, for example, at least about 5% (e.g.,about 10%, about 20%, or about 30%). In some embodiments, the dose ofsclerostin binding agent elevates the level of a bone formation markerat least about 40% (e.g., about 50%, about 60%, or about 70%). In otherembodiments, the dose of sclerostin binding agent increases the level ofone or more bone formation markers by at least about 75% (e.g., about80%, about 90%, about 100%, or about 110%). In yet other embodiments,the dose of sclerostin binding agent increases the level of a boneformation marker by at least about 120% (e.g., about 130%, about 140%,about 150%, about 160% or about 170%). In alternative embodiments, thesclerostin binding agent increases the level of bone formation marker byleast about 180% (e.g., about 190% or about 200%). Bone formation markerlevels ideally remain elevated (compared to bone formation marker levelspre-treatment or to a standard range typical of that patient population)for at least about 3 days (e.g., about 7 days, about 2 weeks, about 3weeks, about 1 month, about 5 weeks, about 6 weeks, about 7 weeks, about2 months, about 9 weeks, about 10 weeks, about 11 weeks, or about 3months) after administration of a single dose of the sclerostin bindingagent.

The invention also provides a method of increasing bone mineral density(BMD), wherein an amount of sclerostin binding agent that (a) reducesthe level of a marker of bone resorption and (b) increases the level ofa marker of bone formation is administered to a human. BMD generallycorrelates with skeletal fragility and osteoporosis. Typically, BMD iscan be measured “total body” (e.g., head, trunk, arms, and legs) or atthe hip (e.g., total hip and/or femoral neck), spine (e.g., lumbarspine), wrist, finger, shin bone and/or heel. In osteoporosis diagnosis,a patient's BMD is compared to the peak density of a 30-year old healthyadult (i.e., a “young adult”), creating the so-called “T-score.” Apatient's BMD also may be compared to an “age-matched” bone density(see, e.g., World Health Organization Scientific Group on the Preventionand Management of Osteoporosis, “Prevention and management ofosteoporosis: report of a WHO scientific group.” WHO Technical ReportSeries; 921, Geneva, Switzerland (2000)). The difference between apatient's BMD and that of a healthy, young adult is conventionallyreferred to in terms of the multiple of a “standard deviation,” whichtypically equals about 10% to about 12% decrease in bone density. TheWorld Health Organization proposed four diagnostic categories based onBMD T-scores. A BMD value within 1 standard deviation of the young adultreference mean (T-score≧−1) is “normal.” Low bone mass (osteopenia) isindicated by a BMD value more than 1 standard deviation below the youngadult mean, but less than 2 standard deviations (T-score<−1 and>−2.5). AT-score of more than 2.5 standard deviations below the norm supports adiagnosis of osteoporosis. If a patient additionally suffers from one ormore fragility fractures, the patient qualifies as having severeosteoporosis.

The sclerostin inhibitor, e.g., a sclerostin binding agent, may beadministered to a patient to improve bone mineral density regardless ofthe patient's T-score. The sclerostin binding agent may be administeredat a dose and for a time period effective to increase BMD in the patientby at least about 1% (about 2%, about 3%, about 4%, about 5%, or about6%). In some embodiments, BMD is increased by at least about 8% (e.g.,at least about 10%, about 12%, about 15%, or about 18%). In otherembodiments, BMD is increased by the sclerostin binding agent at leastabout 20% (e.g., at least about 22%, about 25%, or about 28%) at thehip, spine, wrist, finger, shin bone, and/or heel. In yet otherembodiments, BMD is increased at least about 30% (e.g., at least about32%, about 35%, about 38%, or about 40%). In other words, the BMD can beincreased to the range of about 1 to about 2.5 standard deviations(preferably a range of about 0 to about 1 standard deviations) below thenormal BMD of a healthy young adult.

Alterations in bone remodeling can lead to fluctuations in mineralconcentrations throughout the body. Bone is one of the principalregulators of calcium levels in the bloodstream. Osteoclast-mediatedbone resorption releases stored calcium into the systemic circulation,while osteoblast-mediated bone formation removes calcium fromcirculation to incorporate into bone tissue. In normal bone remodeling,these processes cycle to maintain healthy, strong bone and maintain freecalcium levels at about 8.5 mg/dL to about 10.5 mg/dL (e.g., about 2.2mmol/L to about 2.6 mmol/L). Bone disorders, other illnesses, and evencertain therapies can disrupt systemic calcium levels with direconsequences. Hypercalcemia is associated with high levels of calcium inthe blood (e.g., greater than 12 mg/dL or 3 mmol/L). Extraordinarilyhigh calcium levels leads to, for example, fatigue, confusion,constipation, decreased appetite, frequent urination, heart problems,and bone pain. Hypocalcemia is an electrolyte imbalance indicated by anabnormally low level of calcium in the blood (e.g., less than about 9mg/dL or 2.2 mmol/L). Calcium levels of <7.5 mg/dL (<1.87 mmol/L) orless are considered severe hypocalcemia and may be accompanied byclinical symptoms.

Common symptoms of hypocalcemia include nerve and muscle spasms andcramps, numbness, tingling in the extremities, confusion, and heartirregularities. Extreme variations in system calcium can lead to comaand death.

Several ailments and pharmaceutical therapies alter system calciumlevels. Hypercalcemia and hypocalcemia can result from, for example,chronic kidney disease, renal failure, primary or secondaryhyperparathyroidism, pseudohyperparathyroidism, hypoparathyroidism,pseudohypoparathyroidism, magnesium depletion, alcoholism,bisphosphonate therapy, severe hypermagnesemia, vitamin D deficiency,hyperphosphatemia, acute pancreatitis, hungry bone syndrome, chelation,osteoblastic metastases, sepsis, surgery, chemotherapy, neoplasiasyndrome, familial hypocalciuric hypercalcemia, sarcoidosis,tuberculosis, berylliosis, histoplasmosis, Candidiasis,Coccidioidomycosis, histiocytosis X, Hodgkin's or Non-Hodgkin'slymphoma, Crohn's disease, Wegener's granulomatosis, leukemia,pneumonia, silicone-induced granulomas, immobilization, or drug therapy,such as administration of thiazide diuretics, lithium, estrogens,fluorides, glucose, and insulin. In addition, serum calcium fluctuationsare a side effect of many existing bone-related therapies, such asbisphosphonate and parathyroid hormone therapy. Because of thepotentially life-threatening consequences of calcium imbalance, patientssusceptible to hypocalcemia or hypercalcemia may need to forego certaintherapy options.

Remarkably, sclerostin inhibitors, e.g., sclerostin binding agents, havebeen shown to promote bone formation and inhibit (or slow) boneresorption with minimal fluctuations in systemic calcium levels (e.g.,calcium levels fluctuate 10% or less from baseline serum calciumlevels). Accordingly, the materials and method of the invention areparticularly advantageous in treating patients that are susceptible orsensitive to unstable calcium levels. The amount of sclerostin bindingagent administered to a human in the context of this aspect of theinvention is an amount that does not result in hypocalcemia orhypercalcemia (e.g., clinically-significant hypocalcemia orhypercalcemia). In addition, the invention provides a method of treatinga bone-related disorder in a human suffering from or at risk ofhypocalcemia or hypercalcemia or a human in which treatment withbisphosphonate, a parathyroid hormone, or parathyroid hormone analog iscontraindicated. The method comprises administering to the human anamount of a sclerostin binding agent effective to increase the level ofa marker of bone formation, such as serum levels of BSAP, P1NP, and/orOstCa and/or reduce the level of a marker of bone resorption, such asCTX.

The inventive method is useful for treating or preventing bone-relateddisorders, such as bone-related disorders associated with abnormalosteoblast or osteoclast activity. Indeed, the sclerostin inhibitor(e.g., sclerostin binding agent) can be administered to a humansuffering from a bone related disorder selected from the groupconsisting of achondroplasia, cleidocranial dysostosis,enchondromatosis, fibrous dysplasia, Gaucher's Disease, hypophosphatemicrickets, Marfan's syndrome, multiple hereditary exotoses,neurofibromatosis, osteogenesis imperfecta, osteopetrosis,osteopoikilosis, sclerotic lesions, pseudoarthrosis, pyogenicosteomyelitis, periodontal disease, anti-epileptic drug induced boneloss, primary and secondary hyperparathyroidism, familialhyperparathyroidism syndromes, weightlessness induced bone loss,osteoporosis in men, postmenopausal bone loss, osteoarthritis, renalosteodystrophy, infiltrative disorders of bone, oral bone loss,osteonecrosis of the jaw, juvenile Paget's disease, melorheostosis,metabolic bone diseases, mastocytosis, sickle cell anemia/disease, organtransplant related bone loss, kidney transplant related bone loss,systemic lupus erythematosus, ankylosing spondylitis, epilepsy, juvenilearthritides, thalassemia, mucopolysaccharidoses, Fabry Disease, TurnerSyndrome, Down Syndrome, Klinefelter Syndrome, leprosy, Perthe'sDisease, adolescent idiopathic scoliosis, infantile onset multi-systeminflammatory disease, Winchester Syndrome, Menkes Disease, Wilson'sDisease, ischemic bone disease (such as Legg-Calve-Perthes disease andregional migratory osteoporosis), anemic states, conditions caused bysteroids, glucocorticoid-induced bone loss, heparin-induced bone loss,bone marrow disorders, scurvy, malnutrition, calcium deficiency,osteoporosis, osteopenia, alcoholism, chronic liver disease,postmenopausal state, chronic inflammatory conditions, rheumatoidarthritis, inflammatory bowel disease, ulcerative colitis, inflammatorycolitis, Crohn's disease, oligomenorrhea, amenorrhea, pregnancy,diabetes mellitus, hyperthyroidism, thyroid disorders, parathyroiddisorders, Cushing's disease, acromegaly, hypogonadism, immobilizationor disuse, reflex sympathetic dystrophy syndrome, regional osteoporosis,osteomalacia, bone loss associated with joint replacement, HIVassociated bone loss, bone loss associated with loss of growth hormone,bone loss associated with cystic fibrosis, chemotherapy-associated boneloss, tumor-induced bone loss, cancer-related bone loss, hormoneablative bone loss, multiple myeloma, drug-induced bone loss, anorexianervosa, disease-associated facial bone loss, disease-associated cranialbone loss, disease-associated bone loss of the jaw, disease-associatedbone loss of the skull, bone loss associated with aging, facial boneloss associated with aging, cranial bone loss associated with aging, jawbone loss associated with aging, skull bone loss associated with aging,and bone loss associated with space travel.

The inventive method need not cure the patient of the disorder orcompletely protect against the onset of a bone-related disorder toachieve a beneficial biological response. The method may be usedprophylactically, meaning to protect, in whole or in part, against abone-related disorder or symptom thereof. The method also may be usedtherapeutically to ameliorate, in whole or in part, a bone-relateddisorder or symptom thereof, or to protect, in whole or in part, againstfurther progression of a bone-related disorder or symptom thereof.Indeed, the materials and methods of the invention are particularlyuseful for increasing bone mineral density and maintaining the increasedBMD over a period of time. In this regard, the invention provides amethod of treating a bone-related disorder, which method comprises (a)administering one or more amounts of a sclerostin binding agenteffective to increase BMD measured for the total body (e.g., head,trunk, arms, and legs) or at the hip (e.g., total hip and/or femoralneck), spine (e.g., lumbar spine), wrist, finger, shin bone and/or heelby about 1%, about 2%, about 3%, about 6%, about 8%, about 10%, about12%, about 15%, about 18%, about 20%, about 25%, or 30% or more. One ormore administrations of a pharmaceutical composition comprising thesclerostin binding agent may be carried out over a therapeutic periodof, for example, about 1 month to about 12 months (e.g., about 2 months,about 3 months, about 4 months, about 5 months, about 6 months, about 7months, about 8 months, about 9 months, about 10 months, or about 11months). The method further includes (b) subsequently administering oneor more amounts of a sclerostin binding agent effective to maintain bonemineral density. By “maintain bone mineral density” is meant that theincreased BMD resulting from step (a) does not fall more than about 1%to about 5% over the course of step (b) (e.g., about 6 months, about 9months about 1 year, about 18 months, about 2 years, or over the courseof the patient's life). It will be appreciated that a patient canrequire alternate treatment phases for increasing bone density andmaintaining bone density.

The sclerostin binding agent is preferably administered to a patient ina physiologically-acceptable (e.g., pharmaceutical) composition, whichcan include carriers, excipients, or diluents. It will be appreciatedthat the sclerostin binding agents described herein may be used in thepreparation of a medicament for administration using any of the dosageand timing regimens disclosed herein. Pharmaceutical compositions andmethods of treatment are disclosed in U.S. Patent Publication No.20050106683, which is incorporated by reference herein.“Physiologically-acceptable” refers to molecular entities andcompositions that do not produce an allergic or similar untowardreaction when administered to a human. In addition, the compositionadministered to a subject may contain more than one sclerostin inhibitor(e.g., a sclerostin binding agent and a synthetic chemical sclerostininhibitor) or a sclerostin inhibitor in combination with one or moretherapeutics having different mechanisms of action.

The development of suitable dosing and treatment regimens for using theparticular compositions described herein in a variety of treatmentregimens, including e.g., subcutaneous, oral, parenteral, intravenous,intranasal, and intramuscular administration and formulation, is wellknown in the art and discussed in U.S. Patent Publication No.20070110747. For example, in certain circumstances, it will be desirableto deliver a pharmaceutical composition comprising a sclerostin bindingagent subcutaneously, parenterally, intravenously, intramuscularly, oreven intraperitoneally. Such approaches are well known to the skilledartisan, some of which are further described, for example, in U.S. Pat.Nos. 5,543,158; 5,641,515; and 5,399,363. Illustrative pharmaceuticalforms suitable for injectable use include sterile aqueous solutions ordispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersions (for example, see U.S. Pat.No. 5,466,468). In all cases the form must be sterile and must be fluidto the extent that easy syringability exists.

In one embodiment, for parenteral administration in an aqueous solution,the solution should be suitably buffered if necessary and the liquiddiluent first rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous, and intraperitoneal administration. Forexample, one dose may be dissolved in 1 ml of isotonic NaCl solution andeither added to 1000 ml of hypodermoclysis fluid or injected at theproposed site of infusion (see, for example, Remington's PharmaceuticalSciences, 15th ed., Mack Pub. Co., Easton, Pa., pp. 1035-1038 and1570-1580). Some variation in dosage and frequency of administration mayoccur depending on the condition of the subject being treated; age,height, weight, and overall health of the patient; and the existence ofany side effects. In addition, a pharmaceutical composition comprising asclerostin binding agent may be placed within containers (e.g., vials),along with packaging material that provides instructions regarding theuse of such pharmaceutical compositions. Generally, such instructionswill include a tangible expression describing the reagent concentration,as well as within certain embodiments, relative amounts of excipientingredients or diluents (e.g., water, saline or PBS) that may benecessary to reconstitute the pharmaceutical composition.

The sclerostin binding agent is administered in an amount that reducesthe level of a bone resorption marker and/or increases the level of abone formation marker and/or increases bone density. The dose ofsclerostin binding agent administered may range from about 0.5 mg/kg toabout 20 mg/kg (e.g., 12 mg/kg) of body weight. For example, the dose ofsclerostin binding agent may range from about 1 mg/kg to about 10 mg/kg(e.g., about 2 mg/kg or about 9 mg/kg), about 1 mg/kg to about 3 mg/kg,or about 3 mg/kg to about 8 mg/kg (e.g., about 4 mg/kg, 5 mg/kg, 6mg/kg, or 7 mg/kg).

In addition, it may be advantageous to administer multiple doses of asclerostin binding agent or space out the administration of doses,depending on the therapeutic regimen selected for a particular patient.The sclerostin binding agent can be administered periodically over atime period of one year or less (e.g., 9 months or less, 6 months orless, or 3 months or less). In this regard, the sclerostin binding agentcan be administered to the human once every about 7 days, or 2 weeks, or3 weeks, or 1 month, or 5 weeks, or 6 weeks, or 7 weeks, or 2 months, or9 weeks, or 10 weeks, or 11 weeks, or 3 months, or 13 weeks, or 14weeks, or 15 weeks, or 4 months, or 17 weeks, or 18 weeks, or 19 weeks,or 5 months, or 21 weeks, or 22 weeks, or 23 weeks, or 6 months, or 12months.

The inventive method comprises administering an amount of a “sclerostininhibitor.” As used herein, the term “sclerostin inhibitor” means anymolecule that inhibits the biological activity of sclerostin on bone, asmeasured by changes to bone mineralization, bone density, effect onosteoblasts and/or osteoclasts, markers of bone formation, markers ofbone resorption, markers of osteoblast activity, and/or markers ofosteoclast activity. Such inhibitors may act by binding to sclerostin orits receptor or binding partner. Inhibitors in this category include“sclerostin binding agents,” such as, e.g., antibodies or peptide-basedmolecules. “Sclerostin inhibitors” also refers to small organic chemicalcompounds, optionally of less than about 1000 Daltons in molecularweight that bind sclerostin and inhibit its activity. Inhibitors mayalternatively act by inhibiting expression of sclerostin. Inhibitors inthis category include polynucleotides or oligonucleotides that bind tosclerostin DNA or mRNA and inhibit sclerostin expression, including anantisense oligonucleotide, inhibitory RNA, DNA enzyme, ribozyme, anaptamer or pharmaceutically acceptable salts thereof that inhibit theexpression of sclerostin.

A “sclerostin binding agent” specifically binds to sclerostin orportions thereof to block or impair binding of human sclerostin to oneor more ligands. Sclerostin, the product of the SOST gene, is absent insclerosteosis, a skeletal disease characterized by bone overgrowth andstrong dense bones (Brunkow et al., Am. J. Hum. Genet., 68:577-589(2001); Balemans et al., Hum. Mol. Genet., 10:537-543 (2001)). The aminoacid sequence of human sclerostin is reported by Brunkow et al. and isdisclosed in U.S. Patent Publication No. 20070110747 as SEQ ID NO: 1(which patent publication is incorporated in its entirety for itsdescription of sclerostin binding agents and Sequence Listing).Recombinant human sclerostin/SOST is commercially available from R&DSystems (Minneapolis, Minn., USA; 2006 Catalog #1406-ST-025).Additionally, recombinant mouse sclerostin/SOST is commerciallyavailable from R&D Systems (Minneapolis, Minn., USA; 2006 Catalog#1589-ST-025). Research grade sclerostin-binding monoclonal antibodiesare commercially available from R&D Systems (Minneapolis, Minn., USA;mouse monoclonal: 2006 Catalog # MAB1406; rat monoclonal: 2006 Catalog #MAB1589). U.S. Pat. Nos. 6,395,511 and 6,803,453, and U.S. PatentPublication Nos. 20040009535 and 20050106683 refer to anti-sclerostinantibodies generally. Examples of sclerostin binding agents suitable foruse in the context of the invention also are described in U.S. PatentPublication Nos. 20070110747 and 20070072797, which are herebyincorporated by reference. Additional information regarding materialsand methods for generating sclerostin binding agents can be found inU.S. Patent Publication No. 20040158045.

The sclerostin binding agent of the invention preferably is an antibody.The term “antibody” refers to an intact antibody, or a binding fragmentthereof. An antibody may comprise a complete antibody molecule(including polyclonal, monoclonal, chimeric, humanized, or humanversions having full length heavy and/or light chains), or comprise anantigen binding fragment thereof. Antibody fragments include F(ab′)₂,Fab, Fab′, Fv, Fc, and Fd fragments, and can be incorporated into singledomain antibodies, single-chain antibodies, maxibodies, minibodies,intrabodies, diabodies, triabodies, tetrabodies, v-NAR and bis-scFv(see, e.g., Hollinger and Hudson, Nature Biotechnology, 23(9):1126-1136(2005)). Antibody polypeptides, including fibronectin polypeptidemonobodies, also are disclosed in U.S. Pat. No. 6,703,199. Otherantibody polypeptides are disclosed in U.S. Patent Publication No.20050238646. Anti-sclerostin antibodies may bind to sclerostin of SEQ IDNO: 1, or a naturally occurring variant thereof, with an affinity ofless than or equal to 1×10⁻⁷M, less than or equal to 1×10⁻⁸M, less thanor equal to 1×10⁻⁹M, less than or equal to 1×10⁻¹⁰M, less than or equalto 1×10⁻¹¹M, or less than or equal to 1×10⁻¹² M. Affinity may bedetermined by an affinity ELISA assay. In certain embodiments, affinitymay be determined by a BIAcore assay. In certain embodiments, affinitymay be determined by a kinetic method. In certain embodiments, affinitymay be determined by an equilibrium/solution method.

An antibody fragment may be any synthetic or genetically engineeredprotein. For example, antibody fragments include isolated fragmentsconsisting of the light chain variable region, “Fv” fragments consistingof the variable regions of the heavy and light chains, recombinantsingle chain polypeptide molecules in which light and heavy variableregions are connected by a peptide linker (scFv proteins).

Another form of an antibody fragment is a peptide comprising one or morecomplementarity determining regions (CDRs) of an antibody. CDRs (alsotermed “minimal recognition units” or “hypervariable region”) can beobtained by constructing polynucleotides that encode the CDR ofinterest. Such polynucleotides are prepared, for example, by using thepolymerase chain reaction to synthesize the variable region using mRNAof antibody-producing cells as a template (see, for example, Larrick etal., Methods: A Companion to Methods in Enzymology, 2:106 (1991);Courtenay-Luck, “Genetic Manipulation of Monoclonal Antibodies,” inMonoclonal Antibodies Production, Engineering and Clinical Application,Ritter et al. (eds.), page 166, Cambridge University Press (1995); andWard et al., “Genetic Manipulation and Expression of Antibodies,” inMonoclonal Antibodies: Principles and Applications, Birch et al.,(eds.), page 137, Wiley-Liss, Inc. (1995)).

In one embodiment of the invention, the sclerostin binding agentcross-blocks the binding of at least one of antibodies Ab-A, Ab-B, Ab-C,Ab-D, Ab-1, Ab-2, Ab-3, Ab-4, Ab-5, Ab-6, Ab-7, Ab-8, Ab-9, Ab-10,Ab-11, Ab-12, Ab-13, Ab-14, Ab-15, Ab-16, Ab-17, Ab-18, Ab-19, Ab-20,Ab-21, Ab-22, Ab-23, and Ab-24 (all of which are described in U.S.Patent Publication No. 20070110747) to sclerostin. Alternatively or inaddition, the sclerostin binding agent is cross-blocked from binding tosclerostin by at least one of antibodies Ab-A, Ab-B, Ab-C, Ab-D, Ab-1,Ab-2, Ab-3, Ab-4, Ab-5, Ab-6, Ab-7, Ab-8, Ab-9, Ab-10, Ab-11, Ab-12,Ab-13, Ab-14, Ab-15, Ab-16, Ab-17, Ab-18, Ab-19, Ab-20, Ab-21, Ab-22,Ab-23, and Ab-24 (all of which are described in U.S. Patent PublicationNo. 20070110747). The terms “cross-block,” “cross-blocked,” and“cross-blocking” are used interchangeably herein to mean the ability ofan antibody or other binding agent to interfere with the binding ofother antibodies or binding agents to sclerostin. The extent to which anantibody or other binding agent is able to interfere with the binding ofanother to sclerostin, and therefore whether it can be said tocross-block, can be determined using competition binding assays. In someaspects of the invention, a cross-blocking antibody or fragment thereofreduces sclerostin binding of a reference antibody between about 40% andabout 100%, such as about 60% and about 100%, specifically between 70%and 100%, and more specifically between 80% and 100%. A particularlysuitable quantitative assay for detecting cross-blocking uses a Biacoremachine which measures the extent of interactions using surface plasmonresonance technology. Another suitable quantitative cross-blocking assayuses an ELISA-based approach to measure competition between antibodiesor other binding agents in terms of their binding to sclerostin.

Suitable sclerostin binding agents include antibodies and portionsthereof described in U.S. Patent Publication No. 20070110747, such asone or more of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 asspecifically disclosed therein. At least one of the regions of CDR-H1,CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 may have at least one aminoacid substitution, provided that the binding agent retains the bindingspecificity of the non-substituted CDR. The non-CDR portion of thebinding agent may be a non-protein molecule, wherein the binding agentcross-blocks the binding of an antibody disclosed herein to sclerostinand/or neutralizes sclerostin. The non-CDR portion of the binding agentmay be a non-protein molecule in which the binding agent exhibits asimilar binding pattern to human sclerostin peptides in a humansclerostin peptide epitope competition binding assay as that exhibitedby at least one of antibodies Ab-A, Ab-B, Ab-C, Ab-D, Ab-1, Ab-2, Ab-3,Ab-4, Ab-5, Ab-6, Ab-7, Ab-8, Ab-9, Ab-10, Ab-11, Ab-12, Ab-13, Ab-14,Ab-15, Ab-16, Ab-17, Ab-18, Ab-19, Ab-20, Ab-21, Ab-22, Ab-23, and Ab-24(all of which are described in U.S. Patent Publication No. 20070110747),and/or neutralizes sclerostin. The non-CDR portion of the binding agentmay be composed of amino acids, wherein the binding agent is arecombinant binding protein or a synthetic peptide, and the recombinantbinding protein cross-blocks the binding of an antibody to sclerostinand/or neutralizes sclerostin. The non-CDR portion of the binding agentmay be composed of amino acids, wherein the binding agent is arecombinant binding protein, and the recombinant binding proteinexhibits a similar binding pattern to human sclerostin peptides in thehuman sclerostin peptide epitope competition binding assay (described inU.S. Patent Publication No. 20070110747) as that exhibited by at leastone of the antibodies Ab-A, Ab-B, Ab-C, Ab-D, Ab-1, Ab-2, Ab-3, Ab-4,Ab-5, Ab-6, Ab-7, Ab-8, Ab-9, Ab-10, Ab-11, Ab-12, Ab-13, Ab-14, Ab-15,Ab-16, Ab-17, Ab-18, Ab-19, Ab-20, Ab-21, Ab-22, Ab-23, and Ab-24(described in U.S. Patent Publication No. 20070110747), and/orneutralizes sclerostin. Preferably, the sclerostin binding agent isAb-A, Ab-B, Ab-C, Ab-D, Ab-1, Ab-2, Ab-3, Ab-4, Ab-5, Ab-6, Ab-7, Ab-8,Ab-9, Ab-10, Ab-11, Ab-12, Ab-13, Ab-14, Ab-15, Ab-16, Ab-17, Ab-18,Ab-19, Ab-20, Ab-21, Ab-22, Ab-23, or Ab-24 of U.S. Patent PublicationNo. 20070110747.

In addition, the sclerostin binding agent can comprise at least one CDRsequence having at least 75% identity (e.g., 100% identity) to a CDRselected from SEQ ID NOs: 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 78, 79, 80, 81, 99,100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113,114, 115, 116, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247,248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261,262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275,276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289,290, 291, 292, 293, 294, 295, 296, 297, 298, 351, 352, 353, 358, 359,and 360 disclosed in U.S. Patent Publication No. 20070110747.Preferably, the sclerostin binding agent comprises at least one CDRsequence having at least 75% identity to a CDR selected from SEQ ID NOs:245, 246, 247, 78, 79, 80, 269, 270, 271, 239, 240, and 24, all of whichis described in U.S. Patent Publication No. 20070110747. As described inU.S. Patent Publication No. 20070110747, the sclerostin binding agentcan comprise: a) CDR sequences of SEQ ID NOs:54, 55, and 56 and CDRsequences of SEQ ID NOs:51, 52, and 53; b) CDR sequences of SEQ IDNOs:60, 61, and 62 and CDR sequences of SEQ ID NOs:57, 58, and 59; c)CDR sequences of SEQ ID NOs:48, 49, and 50 and CDR sequences of SEQ IDNOs:45, 46, and 47; d) CDR sequences of SEQ ID NOs:42, 43, and 44 andCDR sequences of SEQ ID NOs:39, 40, and 41; e) CDR sequences of SEQ IDNOs:275, 276, and 277 and CDR sequences of SEQ ID NOs:287, 288, and 289;f) CDR sequences of SEQ ID NOs:278, 279, and 280 and CDR sequences ofSEQ ID NOs:290, 291, and 292; g) CDR sequences of SEQ ID NOs:78, 79, and80 and CDR sequences of SEQ ID NOs: 245, 246, and 247; h) CDR sequencesof SEQ ID NOs:81, 99, and 100 and CDR sequences of SEQ ID NOs:248, 249,and 250; i) CDR sequences of SEQ ID NOs:101, 102, and 103 and CDRsequences of SEQ ID NOs:251, 252, and 253; j) CDR sequences of SEQ IDNOs:104, 105, and 106 and CDR sequences of SEQ ID NOs:254, 255, and 256;k) CDR sequences of SEQ ID NOs:107, 108, and 109 and CDR sequences ofSEQ ID NOs:257, 258, and 259; 1) CDR sequences of SEQ ID NOs:110, 111,and 112 and CDR sequences of SEQ ID NOs:260, 261, and 262; m) CDRsequences of SEQ ID NOs:281, 282, and 283 and CDR sequences of SEQ IDNOs:293, 294, and 295; n) CDR sequences of SEQ ID NOs:113, 114, and 115and CDR sequences of SEQ ID NOs:263, 264, and 265; o) CDR sequences ofSEQ ID NOs:284, 285, and 286 and CDR sequences of SEQ ID NOs:296, 297,and 298; p) CDR sequences of SEQ ID NOs:116, 237, and 238 and CDRsequences of SEQ ID NOs:266, 267, and 268; q) CDR sequences of SEQ IDNOs:239, 240, and 241 and CDR sequences of SEQ ID NOs:269, 270, and 271;r) CDR sequences of SEQ ID NOs:242, 243, and 244 and CDR sequences ofSEQ ID NOs:272, 273, and 274; or s) CDR sequences of SEQ ID NOs:351,352, and 353 and CDR sequences of SEQ ID NOs:358, 359, and 360.

The sclerostin binding agent also can comprise at least one CDR sequencehaving at least 75% identity to a CDR selected from CDR-H1, CDR-H2,CDR-H3, CDR-L1, CDR-L2, and CDR-L3 wherein CDR-H1 has the sequence givenin SEQ ID NO: 245 or SEQ ID NO: 269, CDR-H2 has the sequence given inSEQ ID NO: 246 or SEQ ID NO: 270, CDR-H3 has the sequence given in SEQID NO: 247 or SEQ ID NO: 271, CDR-L1 has the sequence given in SEQ IDNO: 78 or SEQ ID NO: 239, CDR-L2 has the sequence given in SEQ ID NO: 79or SEQ ID NO: 240 and CDR-L3 has the sequence given in SEQ ID NO: 80 orSEQ ID NO 241, all of which is described in U.S. Patent Publication No.20070110747.

Alternatively, the sclerostin binding agent can have a heavy chaincomprising CDR's H1, H2, and H3 and comprising a polypeptide having thesequence provided in SEQ ID NO: 137 or a variant thereof in which saidCDR's are at least 75% identical to SEQ ID NO: 245, 246, and 247,respectively, and a light chain comprising CDR's L1, L2 and L3 andcomprising a polypeptide having the sequence provided in SEQ ID NO: 133or a variant thereof in which said CDR's are at least 75% identical toSEQ ID NO: 78, 79, and 80, respectively (as described in U.S. PatentPublication No. 20070110747).

The sclerostin binding agent may have a heavy chain comprising CDR's H1,H2, and H3 and comprising a polypeptide having the sequence provided inSEQ ID NO: 145 or 392 or a variant thereof in which said CDR's are atleast 75% identical to SEQ ID NO: 245, 246, and 247, respectively, and alight chain comprising CDR's L1, L2, and L3 and comprising a polypeptidehaving the sequence provided in SEQ ID NO: 141 or a variant thereof inwhich said CDR's are at least 75% identical to SEQ ID NO: 78, 79, and80, respectively (as described in U.S. Patent Publication No.20070110747).

The sclerostin binding agent may have a heavy chain comprising CDR's H1,H2, and H3 and comprising a polypeptide having the sequence provided inSEQ ID NO: 335 or a variant thereof in which said CDR's are at least 75%identical to SEQ ID NO: 269, 270, and 271, respectively, and a lightchain comprising CDR's L1, L2, and L3 and comprising a polypeptidehaving the sequence provided in SEQ ID NO: 334 or a variant thereof inwhich said CDR's are at least 75% identical to SEQ ID NO: 239, 240, and241, respectively (as described in U.S. Patent Publication No.20070110747).

Alternatively, the sclerostin binding agent has a heavy chain comprisingCDR's H1, H2, and H3 and comprising a polypeptide having the sequenceprovided in SEQ ID NO: 331 or a variant thereof in which said CDR's areat least 75% identical to SEQ ID NO: 269, 270, and 271, respectively,and a light chain comprising CDR's L1, L2, and L3 and comprising apolypeptide having the sequence provided in SEQ ID NO: 330 or a variantthereof in which said CDR's are at least 75% identical to SEQ ID NO:239, 240, and 241, respectively (as described in U.S. Patent PublicationNo. 20070110747).

The sclerostin binding agent may have a heavy chain comprising CDR's H1,H2, and H3 and comprising a polypeptide having the sequence provided inSEQ ID NO: 345 or 396 or a variant thereof in which said CDR's are atleast 75% identical to SEQ ID NO: 269, 270, and 271, respectively, and alight chain comprising CDR's L1, L2, and L3 and comprising a polypeptidehaving the sequence provided in SEQ ID NO: 341 or a variant thereof inwhich said CDR's are at least 75% identical to SEQ ID NO: 239, 240, and241, respectively (as described in U.S. Patent Publication No.20070110747).

Alternatively, the sclerostin binding agent has a heavy chain comprisinga polypeptide having the sequence provided in SEQ ID NO: 137, and alight chain comprising a polypeptide having the sequence provided in SEQID NO: 133; or a heavy chain comprising a polypeptide having thesequence provided in SEQ ID NO: 145 or 392, and a light chain comprisinga polypeptide having the sequence provided in SEQ ID NO: 141; or a heavychain comprising a polypeptide having the sequence provided in SEQ IDNO: 335, and a light chain comprising a polypeptide having the sequenceprovided in SEQ ID NO: 334; or a heavy chain comprising a polypeptidehaving the sequence provided in SEQ ID NO: 331, and a light chaincomprising a polypeptide having the sequence provided in SEQ ID NO: 330;or a heavy chain comprising a polypeptide having the sequence providedin SEQ ID NO: 345 or 396, and a light chain comprising a polypeptidehaving the sequence provided in SEQ ID NO: 341 (as described in U.S.Patent Publication No. 20070110747).

Sclerostin binding agents for use in the inventive method preferablymodulate sclerostin function in the cell-based assay described in U.S.Patent Publication No. 20070110747 and/or the in vivo assay described inU.S. Patent Publication No. 20070110747 and/or bind to one or more ofthe epitopes described in U.S. Patent Publication No. 20070110747 and/orcross-block the binding of one of the antibodies described in U.S.Patent Publication No. 20070110747 and/or are cross-blocked from bindingsclerostin by one of the antibodies described in U.S. Patent PublicationNo. 20070110747.

Alternatively, the inventive method can comprise administering asclerostin inhibitor other than a sclerostin binding agent describedherein. Such agents can act directly or indirectly on SOST orsclerostin. Sclerostin inhibitors contemplated for use in the inventivemethod include those described in U.S. Patent Publication No.20030229041 (the entire disclosure of which is hereby incorporated byreference, with particular emphasis upon the description of sclerostininhibitors). For example, agents useful for modulating SOST expressionand sclerostin activity include, but are not limited to, steroids (suchas those corresponding to Formula 1 of U.S. Patent Publication No.20030229041), alkaloids, terpenoids, peptoids, and synthetic chemicals.In some embodiments, the SOST antagonist or agonist can bind to aglucocorticoid receptor. For example, dexamethasone tends to abolish thestimulatory effect of BMP-4 and BMP-6 on SOST expression. Other chemicalentities including glucocorticoid analogs, bile salts (such as thosecorresponding to Formula 3 of U.S. Patent Publication No. 20030229041),and prostaglandins (such as those corresponding to Formula 2 of U.S.Patent Publication No. 20030229041) also modulate the effects of bonemorphogenetic proteins on SOST expression, and are contemplated for usein the inventive method.

The sclerostin inhibitor may also be other small molecule therapeuticsthat act directly or indirectly on SOST or sclerostin to decrease thelevel of at least one bone resorptive marker and/or increase the levelof at least one bone formation marker in vivo. The term “small molecule”includes a compound or molecular complex, either synthetic, naturallyderived, or partially synthetic, and which preferably has a molecularweight of less than 5,000 Daltons (e.g., between about 100 and 1,500Daltons). Agents can be obtained using any of the numerous approaches incombinatorial library methods known in the art, including spatiallyaddressable parallel solid phase or solution phase libraries, syntheticlibrary methods requiring deconvolution, the “one-bead one-compound”library method, and synthetic library methods using affinitychromatography selection (see, e.g., Lam, Anticancer Drug Des., 12:145(1997) and U.S. Pat. Nos. 5,738,996; 5,807,683; and 7,261,892). Methodsof developing and screening sclerostin inhibitors are further describedin U.S. Patent Publication No. 20030229041, the discussion of which ishereby incorporated by reference.

Sclerostin expression inhibitors that may be used according to themethods of the invention include inhibitor oligonucleotides orpolynucleotides, including pharmaceutically acceptable salts thereof,e.g., sodium salts. Nonlimiting examples include: antisenseoligonucleotides (Eckstein, Antisense Nucleic Acid Drug Dev., 10:117-121 (2000); Crooke, Methods Enzymol., 313: 3-45 (2000); Guvakova etal., J. Biol. Chem., 270: 2620-2627 (1995); Manoharan, Biochim. Biophys.Acta, 1489: 117-130 (1999); Baker et al., J. Biol. Chem., 272:11994-12000 (1997); Kurreck, Eur. J. Biochem., 270: 1628-1644 (2003);Sierakowska et al., Proc. Natl. Acad. Sci. USA, 93: 12840-12844 (1996);Marwick, J. Am. Med. Assoc., 280: 871 (1998); Tomita and Morishita,Curr. Pharm. Des., 10: 797-803 (2004); Gleave and Monia, Nat. Rev.Cancer, 5: 468-479 (2005) and Patil, AAPS J., 7: E61-E77 (2005)),triplex oligonucleotides (Francois et al., Nucleic Acids Res., 16:11431-11440 (1988) and Moser and Dervan, Science, 238: 645-650 (1987)),ribozymes/deoxyribozymes (DNAzymes) (Kruger et al., Tetrahymena. Cell,31: 147-157 (1982); Uhlenbeck, Nature, 328: 596-600 (1987); Sigurdssonand Eckstein, Trends Biotechnol., 13: 286-289 (1995); Kumar et al., GeneTher., 12: 1486-1493 (2005); Breaker and Joyce, Chem. Biol., 1: 223-229(1994); Khachigian, Curr. Pharm. Biotechnol., 5: 337-339 (2004);Khachigian, Biochem. Pharmacol., 68: 1023-1025 (2004) and Trulzsch andWood, J. Neurochem., 88: 257-265 (2004)), small-interfering RNAs/RNAi(Fire et al., Nature, 391: 806-811 (1998); Montgomery et al., Proc.Natl. Acad. Sci. U.S.A., 95: 15502-15507 (1998); Cullen, Nat. Immunol.,3: 597-599 (2002); Hannon, Nature, 418: 244-251 (2002); Bernstein etal., Nature, 409: 363-366 (2001); Nykanen et al., Cell, 107: 309-321(2001); Gilmore et al., J. Drug Target., 12: 315-340 (2004); Reynolds etal., Nat. Biotechnol., 22: 326-330 (2004); Soutschek et al., Nature,432173-178 (2004); Ralph et al., Nat. Med., 11: 429-433 (2005); Xia etal., Nat. Med., 10816-820 (2004) and Miller et al., Nucleic Acids Res.,32: 661-668 (2004)), aptamers (Ellington and Szostak, Nature, 346:818-822 (1990); Doudna et al., Proc. Natl. Acad. Sci. U.S.A., 92:2355-2359 (1995); Tuerk and Gold, Science, 249: 505-510 (1990); White etal., Mol. Ther., 4: 567-573 (2001); Rusconi et al., Nature, 419: 90-94(2002); Nimjee et al., Mol. Ther., 14: 408-415 (2006); Gragoudas et al.,N. Engl. J. Med., 351: 3805-2816 (2004); Vinores, Curr. Opin. Mol.Ther., 5673-679 (2003) and Kourlas and Schiller et al., Clin. Ther., 28:36-44 (2006)) or decoy oligonucleotides (Morishita et al., Proc. Natl.Acad. Sci. U.S.A., 92: 5855-5859 (1995); Alexander et al., J. Am. Med.Assoc., 294: 2446-2454 (2005); Mann and Dzau, J. Clin. Invest., 106:1071-1075 (2000) and Nimjee et al., Annu. Rev. Med., 56: 555-583(2005)). The foregoing documents are hereby incorporated by reference intheir entirety herein, with particular emphasis on those sections of thedocuments relating to methods of designing, making and using inhibitoryoligonucleotides. Commercial providers such as Ambion Inc. (Austin,Tex.), Darmacon Inc. (Lafayette, Colo.), InvivoGen (San Diego, Calif.),and Molecular Research Laboratories, LLC (Herndon, Va.) generate customsiRNA molecules. In addition, commercial kits are available to producecustom siRNA molecules, such as SILENCER™ siRNA Construction Kit (AmbionInc., Austin, Tex.) or psiRNA System (InvivoGen, San Diego, Calif.).

Inhibitory oligonucleotides which are stable, have a high resistance tonucleases, possess suitable pharmacokinetics to allow them to traffic totarget tissue site at non-toxic doses, and have the ability to crossthrough plasma membranes are contemplated for use as a therapeutic.Inhibitory oligonucleotides may be complementary to the coding portionof a target gene, 3′ or 5′ untranslated regions, or intronic sequencesin a gene, or alternatively coding or intron sequences in the targetmRNA. Intron sequences are generally less conserved and thus may providegreater specificity. In one embodiment, the inhibitory oligonucleotideinhibits expression of a gene product of one species but not itshomologue in another species; in other embodiments, the inhibitoryoligonucleotide inhibits expression of a gene in two species, e.g. humanand primate, or human and murine.

The constitutive expression of antisense oligonucleotides in cells hasbeen shown to inhibit gene expression, possibly via the blockage oftranslation or prevention of splicing. In certain embodiments, theinhibitory oligonucleotide is capable of hybridizing to at least 8, 9,10, 11, or 12 consecutive bases of the sclerostin gene or mRNA (or thereverse strand thereof) under moderate or high stringency conditions.Suitable inhibitory oligonucleotides may be single stranded and containa segment, e.g. at least 12, 15 or 18 bases in length, that issufficiently complementary to, and specific for, an mRNA or DNA moleculesuch that it hybridizes to the mRNA or DNA molecule and inhibitstranscription, splicing or translation. Generally complementarity over alength of less than 30 bases is more than sufficient.

Typically, stringent conditions will be those in which the saltconcentration is less than about 1.5 M Na ion, typically about 0.01 to1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and thetemperature is at least about 30° C. for short nucleic acids (e.g., 10to 50 nucleotides) and at least about 60° C. for longer nucleic acids(e.g., greater than 50 nucleotides). Stringent conditions may also beachieved with the addition of destabilizing agents such as formamide.Exemplary low stringency conditions include hybridization with a buffersolution of 30% to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecylsulphate) at 37° C., and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 Mtrisodium citrate) at 50° C. to 55° C. Exemplary moderate stringencyconditions include hybridization in 40% to 45% formamide, 1.0 M NaCl, 1%SDS at 37° C., and a wash in 0.5× to 1×SSC at 55° C. to 60° C. Exemplaryhigh stringency conditions include hybridization in 50% formamide, 1 MNaCl, 1% SDS at 37° C., and a wash in 0.1×SSC at 60° C. to 65° C.Duration of hybridization is generally less than about 24 hours, usuallyabout 4 hours to about 12 hours.

In some cases, depending on the length of the complementary region, one,two or more mismatches may be tolerated without affecting inhibitoryfunction. In certain embodiments, the inhibitory oligonucleotide is anantisense oligonucleotide, an inhibitory RNA (including siRNA or RNAi,or shRNA), a DNA enzyme, a ribozyme (optionally a hammerhead ribozyme),an aptamer, or pharmaceutically acceptable salts thereof. In oneembodiment, the oligonucleotide is complementary to at least 10 bases ofthe nucleotide sequence encoding SEQ ID NO: 1 of U.S. Patent PublicationNo. 20040158045. In one embodiment, the oligonucleotide targets thenucleotides located in the vicinity of the 3′ untranslated region of thesclerostin mRNA.

The specific sequence utilized in design of the oligonucleotides may beany contiguous sequence of nucleotides contained within the expressedgene message of the target. Factors that govern a target site for theinhibitory oligonucleotide sequence include the length of theoligonucleotide, binding affinity, and accessibility of the targetsequence. Sequences may be screened in vitro for potency of theirinhibitory activity by measuring inhibition of target proteintranslation and target related phenotype, e.g., inhibition of cellproliferation in cells in culture. In general it is known that mostregions of the RNA (5′ and 3′ untranslated regions, AUG initiation,coding, splice junctions and introns) can be targeted using antisenseoligonucleotides. Programs and algorithms, known in the art, may be usedto select appropriate target sequences. In addition, optimal sequencesmay be selected utilizing programs designed to predict the secondarystructure of a specified single stranded nucleic acid sequence andallowing selection of those sequences likely to occur in exposed singlestranded regions of a folded mRNA. Methods and compositions fordesigning appropriate oligonucleotides may be found, for example, inU.S. Pat. No. 6,251,588, the contents of which are incorporated hereinby reference in its entirety.

Phosphorothioate antisense oligonucleotides may be used. Modificationsof the phosphodiester linkage as well as of the heterocycle or the sugarmay provide an increase in efficiency. Phophorothioate is used to modifythe phosphodiester linkage. An N3′-P5′ phosphoramidate linkage has beendescribed as stabilizing oligonucleotides to nucleases and increasingthe binding to RNA. Peptide nucleic acid (PNA) linkage is a completereplacement of the ribose and phosphodiester backbone and is stable tonucleases, increases the binding affinity to RNA, and does not allowcleavage by RNAse H. Its basic structure is also amenable tomodifications that may allow its optimization as an antisense component.With respect to modifications of the heterocycle, certain heterocyclemodifications have proven to augment antisense effects withoutinterfering with RNAse H activity. An example of such modification isC-5 thiazole modification. Finally, modification of the sugar may alsobe considered. 2′-O-propyl and 2′-methoxyethoxy ribose modificationsstabilize oligonucleotides to nucleases in cell culture and in vivo.

Most mRNAs have been shown to contain a number of secondary and tertiarystructures. Secondary structural elements in RNA are formed largely byWatson-Crick type interactions between different regions of the same RNAmolecule. Important secondary structural elements include intramoleculardouble stranded regions, hairpin loops, bulges in duplex RNA andinternal loops. Tertiary structural elements are formed when secondarystructural elements come in contact with each other or with singlestranded regions to produce a more complex three dimensional structure.A number of researchers have measured the binding energies of a largenumber of RNA duplex structures and have derived a set of rules whichcan be used to predict the secondary structure of RNA (see, e.g., Jaegeret al., Proc. Natl. Acad. Sci. USA, 86:7706 (1989); and Turner et al.,Annu. Rev. Biophys. Biophys. Chem. 17:167 (1988)). The rules are usefulin identification of RNA structural elements and, in particular, foridentifying single stranded RNA regions which may represent segments ofthe mRNA to target for siRNA, ribozyme, or antisense technologies.

Short interfering (si) RNA technology (also known as RNAi) generallyinvolves degradation of an mRNA of a particular sequence induced bydouble-stranded RNA (dsRNA) that is homologous to that sequence, thereby“interfering” with expression of the corresponding gene. Any selectedgene may be repressed by introducing a dsRNA which corresponds to all ora substantial part of the mRNA for that gene. It appears that when along dsRNA is expressed, it is initially processed by a ribonuclease IIIinto shorter dsRNA oligonucleotides of as few as 21 to 22 base pairs inlength. Accordingly, siRNA may be affected by introduction or expressionof relatively short homologous dsRNAs. Exemplary siRNAs have sense andantisense strands of about 21 nucleotides that form approximately 19nucleotides of double stranded RNA with overhangs of two nucleotides ateach 3′ end. Indeed the use of relatively short homologous dsRNAs mayhave certain advantages.

Mammalian cells have at least two pathways that are affected bydouble-stranded RNA (dsRNA). In the sequence-specific siRNA pathway, theinitiating dsRNA is first broken into short interfering RNAs, asdescribed above. Short interfering RNAs are thought to provide thesequence information that allows a specific messenger RNA to be targetedfor degradation. In contrast, the nonspecific pathway is triggered bydsRNA of any sequence, as long as it is at least about 30 base pairs inlength.

The nonspecific effects occur because dsRNA activates two enzymes: PKR,which in its active form phosphorylates the translation initiationfactor eIF2 to shut down all protein synthesis, and 2′, 5′oligoadenylate synthetase (2′, 5′-AS), which synthesizes a molecule thatactivates RNase L, a nonspecific enzyme that targets all mRNAs. Thenonspecific pathway may represent a host response to stress or viralinfection, and, in general, the effects of the nonspecific pathway arepreferably minimized. Significantly, longer dsRNAs appear to be requiredto induce the nonspecific pathway and, accordingly, dsRNAs shorter thanabout 30 bases pairs are contemplated to effect gene repression by RNAi(see Hunter et al., J. Biol. Chem., 250: 409-17 (1975); Manche et al.,Mol. Cell. Biol. 12: 5239-48 (1992); Minks et al., J. Biol. Chem., 254:10180-3 (1979); and Elbashir et al., Nature, 411: 494-8 (2001)).

siRNA has proven to be an effective means of decreasing gene expressionin a variety of cell types. siRNA typically decreases expression of agene to lower levels than that achieved using antisense techniques, andfrequently eliminates expression entirely (see Bass, Nature, 411: 428-9(2001)). In mammalian cells, siRNAs are effective at concentrations thatare several orders of magnitude below the concentrations typically usedin antisense experiments (Elbashir et al., Nature, 411:494-8 (2001)).

The double stranded oligonucleotides used to effect RNAi are preferablyless than 30 base pairs in length, for example, about 25, 24, 23, 22,21, 20, 19, 18, or 17 base pairs or less in length, and contain asegment sufficiently complementary to the target mRNA to allowhybridization to the target mRNA. Optionally the dsRNA oligonucleotidesmay include 3′ overhang ends. Exemplary 2-nucleotide 3′ overhangs may becomposed of ribonucleotide residues of any type and may even be composedof 2′-deoxythymidine resides, which lowers the cost of RNA synthesis andmay enhance nuclease resistance of siRNAs in the cell culture medium andwithin transfected cells (see Elbashi et al., supra). Exemplary dsRNAsmay be synthesized chemically or produced in vitro or in vivo usingappropriate expression vectors (see, e.g., Elbashir et al., Genes Dev.,15:188-200 (2001)). Longer RNAs may be transcribed from promoters, suchas T7 RNA polymerase promoters, known in the art.

Longer dsRNAs of 50, 75, 100, or even 500 base pairs or more also may beutilized in certain embodiments of the invention. Exemplaryconcentrations of dsRNAs for effecting RNAi are about 0.05 nM, 0.1 nM,0.5 nM, 1.0 nM, 1.5 nM, 25 nM, or 100 nM, although other concentrationsmay be utilized depending upon the nature of the cells treated, the genetarget and other factors readily discernable to the skilled artisan.

Further compositions, methods and applications of siRNA technology areprovided in U.S. Pat. Nos. 6,278,039; 5,723,750; and 5,244,805, whichare incorporated herein by reference in its entirety.

Compared to siRNA, shRNA offers advantages in silencing longevity anddelivery options. See, e.g., Hannon et al., Nature, 431:371-378 (2004)for review. Vectors that produce shRNAs, which are processedintracellularly into short duplex RNAs having siRNA-like properties havebeen reported (Brummelkamp et al., Science, 296: 550-553 (2000);Paddison et al., Genes Dev., 16: 948-958 (2002)). Such vectors provide arenewable source of a gene-silencing reagent that can mediate persistentgene silencing after stable integration of the vector into the host-cellgenome. Furthermore, the core silencing ‘hairpin’ cassette can bereadily inserted into retroviral, lentiviral, or adenoviral vectors,facilitating delivery of shRNAs into a broad range of cell types(Brummelkamp et al., Cancer Cell, 2:243-247 (2002); Dirac et al., J.Biol. Chem., 278:11731-11734 (2003); Michiels et al., Nat. Biotechnol.,20:1154-1157 (2002); Stegmeie et al., Proc. Natl. Acad. Sci. USA,102:13212-13217 (2005); Khvorova et al., Cell, 115:209-216 (2003)) inany of the innumerable ways that have been devised for delivery of DNAconstructs that allow ectopic mRNA expression.

A hairpin can be organized in either a left-handed hairpin (i.e.,5′-antisense-loop-sense-3′) or a right-handed hairpin (i.e.,5′-sense-loop-antisense-3′). The siRNA may also contain overhangs ateither the 5′ or 3′ end of either the sense strand or the antisensestrand, depending upon the organization of the hairpin. Preferably, ifthere are any overhangs, they are on the 3′ end of the hairpin andcomprise between 1 to 6 bases. The overhangs can be unmodified, or cancontain one or more specificity or stabilizing modifications, such as ahalogen or O-alkyl modification of the 2′ position, or internucleotidemodifications such as phosphorothioate, phosphorodithioate, ormethylphosphonate modifications. The overhangs can be ribonucleic acid,deoxyribonucleic acid, or a combination of ribonucleic acid anddeoxyribonucleic acid.

Additionally, a hairpin can further comprise a phosphate group on the5′-most nucleotide. The phosphorylation of the 5′-most nucleotide refersto the presence of one or more phosphate groups attached to the 5′carbon of the sugar moiety of the 5′-terminal nucleotide. Preferably,there is only one phosphate group on the 5′ end of the region that willform the antisense strand following Dicer processing. In one exemplaryembodiment, a right-handed hairpin can include a 5′ end (i.e., the free5′ end of the sense region) that does not have a 5′ phosphate group, orcan have the 5′ carbon of the free 5′-most nucleotide of the senseregion being modified in such a way that prevents phosphorylation. Thiscan be achieved by a variety of methods including, but not limited to,addition of a phosphorylation blocking group (e.g., a 5′-O-alkyl group),or elimination of the 5′-OH functional group (e.g., the 5′-mostnucleotide is a 5′-deoxy nucleotide). In cases where the hairpin is aleft-handed hairpin, preferably the 5′ carbon position of the 5′-mostnucleotide is phosphorylated.

Hairpins that have stem lengths longer than 26 base pairs can beprocessed by Dicer such that some portions are not part of the resultingsiRNA that facilitates mRNA degradation. Accordingly the first region,which may comprise sense nucleotides, and the second region, which maycomprise antisense nucleotides, may also contain a stretch ofnucleotides that are complementary (or at least substantiallycomplementary to each other), but are or are not the same as orcomplementary to the target mRNA. While the stem of the shRNA can becomposed of complementary or partially complementary antisense and sensestrands exclusive of overhangs, the shRNA can also include thefollowing: (1) the portion of the molecule that is distal to theeventual Dicer cut site contains a region that is substantiallycomplementary/homologous to the target mRNA; and (2) the region of thestem that is proximal to the Dicer cut site (i.e., the region adjacentto the loop) is unrelated or only partially related (e.g.,complementary/homologous) to the target mRNA. The nucleotide content ofthis second region can be chosen based on a number of parametersincluding but not limited to thermodynamic traits or profiles.

Modified shRNAs can retain the modifications in the post-Dicer processedduplex. In exemplary embodiments, in cases in which the hairpin is aright handed hairpin (e.g., 5′-S-loop-AS-3′) containing 2-6 nucleotideoverhangs on the 3′ end of the molecule, 2′-O-methyl modifications canbe added to nucleotides at position 2, positions 1 and 2, or positions1, 2, and 3 at the 5′ end of the hairpin. Also, Dicer processing ofhairpins with this configuration can retain the 5′ end of the sensestrand intact, thus preserving the pattern of chemical modification inthe post-Dicer processed duplex. Presence of a 3′ overhang in thisconfiguration can be particularly advantageous since blunt endedmolecules containing the prescribed modification pattern can be furtherprocessed by Dicer in such a way that the nucleotides carrying the 2′modifications are removed. In cases where the 3′ overhang ispresent/retained, the resulting duplex carrying the sense-modifiednucleotides can have highly favorable traits with respect to silencingspecificity and functionality. Examples of exemplary modificationpatterns are described in detail in U.S. Patent Publication No.20050223427 and International Patent Publication Nos. WO 2004/090105 andWO 2005/078094, the disclosures of each of which are incorporated byreference herein in their entirety.

shRNA may comprise sequences that were selected at random, or accordingto any rational design selection procedure. For example, rational designalgorithms are described in International Patent Publication No. WO2004/045543 and U.S. Patent Publication No. 20050255487, the disclosuresof which are incorporated herein by reference in their entireties.Additionally, it may be desirable to select sequences in whole or inpart based on average internal stability profiles (“AISPs”) or regionalinternal stability profiles (“RISPs”) that may facilitate access orprocessing by cellular machinery.

Ribozymes are enzymatic RNA molecules capable of catalyzing specificcleavage of mRNA, thus preventing translation. (For a review, see Rossi,Current Biology, 4:469-471 (1994)). The mechanism of ribozyme actioninvolves sequence specific hybridization of the ribozyme molecule tocomplementary target RNA, followed by an endonucleolytic cleavage event.The ribozyme molecules preferably include (1) one or more sequencescomplementary to a target mRNA, and (2) the well known catalyticsequence responsible for mRNA cleavage or a functionally equivalentsequence (see, e.g., U.S. Pat. No. 5,093,246, which is incorporatedherein by reference in its entirety).

While ribozymes that cleave mRNA at site-specific recognition sequencescan be used to destroy target mRNAs, hammerhead ribozymes mayalternatively be used. Hammerhead ribozymes cleave mRNAs at locationsdictated by flanking regions that form complementary base pairs with thetarget mRNA. Preferably, the target mRNA has the following sequence oftwo bases: 5′-UG-3′. The construction and production of hammerheadribozymes is well known in the art and is described more fully inHaseloff and Gerlach, Nature, 334:585-591 (1988); and InternationalPatent Publication. No. WO 89/05852, the contents of which areincorporated herein by reference in its entirety.

Gene targeting ribozymes may contain a hybridizing region complementaryto two regions of a target mRNA, each of which is at least 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 contiguous nucleotides(but which need not both be the same length).

Hammerhead ribozyme sequences can be embedded in a stable RNA such as atransfer RNA (tRNA) to increase cleavage efficiency in vivo (Perriman etal., Proc. Natl. Acad. Sci. USA, 92:6175-79 (1995); de Feyter andGaudron, Methods in Molecular Biology, Vol. 74, Chapter 43, “ExpressingRibozymes in Plants,” Turner, P. C. (ed.), Humana Press Inc., Totowa,N.J.). In particular, RNA polymerase III-mediated expression of tRNAfusion ribozymes are well known in the art (see Kawasaki et al., Nature,393:284-9 (1998); Kuwabara et al., Nature Biotechnol., 16:961-5 (1998);and Kuwabara et al., Mol. Cell, 2:617-27 (1998); Koseki et al., J.Virol., 73:1868-77 (1999); Kuwabara et al., Proc. Natl. Acad. Sci. USA,96:1886-91 (1999); Tanabe et al., Nature, 406:473-4 (2000)). There aretypically a number of potential hammerhead ribozyme cleavage siteswithin a given target cDNA sequence. Preferably the ribozyme isengineered so that the cleavage recognition site is located near the 5′end of the target mRNA- to increase efficiency and minimize theintracellular accumulation of non-functional mRNA transcripts.Furthermore, the use of any cleavage recognition site located in thetarget sequence encoding different portions of the target mRNA wouldallow the selective targeting of one or the other target genes.

Ribozymes for use in the inventive method also include RNAendoribonucleases (“Cech-type ribozymes”) such as the one which occursnaturally in Tetrahymena thermophila (known as the IVS, or L-19 IVS RNA)and which has been extensively described in Zaug et al., Science,224:574-578 (1984); Zaug, et al., Science, 231:470-475 (1986); Zaug etal., Nature, 324:429-433 (1986); International Patent Publication No. WO88/04300; and Been et al., Cell, 47:207-216 (1986)). The Cech-typeribozymes have an eight base pair active site which hybridizes to atarget RNA sequence whereafter cleavage of the target RNA takes place.In one embodiment, the inventive method employs those Cech-typeribozymes which target eight base-pair active site sequences that arepresent in a target gene or nucleic acid sequence.

Ribozymes can be composed of modified oligonucleotides (e.g., forimproved stability, targeting, etc.) and can be chemically synthesizedor produced through an expression vector. Because ribozymes, unlikeantisense molecules, are catalytic, a lower intracellular concentrationis required for efficiency. Additionally, in certain embodiments, aribozyme may be designed by first identifying a sequence portionsufficient to cause effective knockdown by RNAi. Portions of the samesequence may then be incorporated into a ribozyme.

Alternatively, target gene expression can be reduced by targetingdeoxyribonucleotide sequences complementary to the regulatory region ofthe gene (i.e., the promoter and/or enhancers) to form triple helicalstructures that prevent transcription of the gene in target cells in thebody. (See generally Helene, C., Anticancer Drug Des., 6:569-84 (1991);Helene et al., Ann. N.Y. Acad. Sci., 660:27-36 (1992); and Maher, L. J.,Bioassays, 14:807-15 (1992)).

Nucleic acid molecules to be used in triple helix formation for theinhibition of transcription are preferably single stranded and composedof deoxyribonucleotides. The base composition of these oligonucleotidesshould promote triple helix formation via Hoogsteen base pairing rules,which generally require sizable stretches of either purines orpyrimidines to be present on one strand of a duplex. Nucleotidesequences may be pyrimidine-based, which will result in TAT and CGCtriplets across the three associated strands of the resulting triplehelix. The pyrimidine-rich molecules provide base complementarity to apurine-rich region of a single strand of the duplex in a parallelorientation to that strand. In addition, nucleic acid molecules may bechosen that are purine-rich, for example, containing a stretch of Gresidues. These molecules will form a triple helix with a DNA duplexthat is rich in GC pairs, in which the majority of the purine residuesare located on a single strand of the targeted duplex, resulting in CGCtriplets across the three strands in the triplex.

Alternatively, the target sequences that can be targeted for triplehelix formation may be increased by creating a so-called “switchback”nucleic acid molecule. Switchback molecules are synthesized in analternating 5′-3′, 3′-5′ manner, such that they base pair with first onestrand of a duplex and then the other, eliminating the necessity for asizable stretch of either purines or pyrimidines to be present on onestrand of a duplex.

Alternatively, DNA enzymes may be used to inhibit expression of targetgene, such as the sclerostin gene. DNA enzymes incorporate some of themechanistic features of both antisense and ribozyme technologies. DNAenzymes are designed so that they recognize a particular target nucleicacid sequence, much like an antisense oligonucleotide. They are,however, also catalytic and specifically cleave the target nucleic acid.

DNA enzymes include two basic types identified by Santoro and Joyce(see, for example, U.S. Pat. No. 6,110,462). The 10-23 DNA enzymecomprises a loop structure which connect two arms. The two arms providespecificity by recognizing the particular target nucleic acid sequencewhile the loop structure provides catalytic function under physiologicalconditions.

Preferably, the unique or substantially unique sequence is a G/C richsegment of approximately 18 to 22 nucleotides. High G/C content helpsinsure a stronger interaction between the DNA enzyme and the targetsequence. The specific antisense recognition sequence that will targetthe enzyme to the message may be divided between the two arms of the DNAenzyme.

Methods of making and administering DNA enzymes can be found, forexample, in U.S. Pat. No. 6,110,462. Additionally, one of skill in theart will recognize that, like antisense oligonucleotide, DNA enzymes canbe optionally modified to improve stability and improve resistance todegradation.

Inhibitory oligonucleotides can be administered directly or delivered tocells by transformation or transfection via a vector, including viralvectors or plasmids, into which has been placed DNA encoding theinhibitory oligonucleotide with the appropriate regulatory sequences,including a promoter, to result in expression of the inhibitoryoligonucleotide in the desired cell. Known methods include standardtransient transfection, stable transfection and delivery using virusesranging from retroviruses to adenoviruses. Delivery of nucleic acidinhibitors by replicating or replication-deficient vectors iscontemplated. Expression can also be driven by either constitutive orinducible promoter systems (Paddison et al., Methods Mol. Biol.,265:85-100 (2004)). In other embodiments, expression may be under thecontrol of tissue or development-specific promoters.

For example, vectors may be introduced by transfection using carriercompositions such as Lipofectamine 2000 (Life Technologies) orOligofectamine (Life Technologies). Transfection efficiency may bechecked using fluorescence microscopy for mammalian cell lines afterco-transfection of hGFP-encoding pAD3 (Kehlenback et al., J. Cell Biol.,141:863-74 (1998)).

The delivery route will be the one that provides the best inhibitoryeffect as measured according to the criteria described above. Deliverymediated by cationic liposomes, delivery by retroviral vectors anddirect delivery are efficient.

The effectiveness of the inhibitory oligonucleotide may be assessed byany of a number of assays, including reverse transcriptase polymerasechain reaction or Northern blot analysis to determine the level ofexisting human sclerostin mRNA, or Western blot analysis usingantibodies which recognize the human sclerostin protein, aftersufficient time for turnover of the endogenous pool after new proteinsynthesis is repressed.

The invention is further described in the following example. The exampleserves only to illustrate the invention and are not intended to limitthe scope of the invention in any way.

EXAMPLE

This example describes in vivo studies wherein a sclerostin bindingagent reduced the level of a marker of bone resorption and increased thelevel of one or more markers of bone formation.

A single-center, randomized, double-blind, placebo-controlled, ascendingsingle-dose study in healthy men and postmenopausal women was conducted.Approximately 72 subjects enrolled in one of six dose cohorts. Forcohorts 1, 2, 3a, 4, 5 and 6a, eight healthy postmenopausal women wererandomized to receive a sclerostin binding agent or placebo viasubcutaneous injection in a 3:1 ratio at dose levels of 0.1 mg/kg, 0.3mg/kg, 1 mg/kg, 3 mg/kg, 5 mg/kg, or 10 mg/kg, respectively. In cohorts3b and 6b, 8 healthy males received the sclerostin binding agent or aplacebo intravenously and subcutaneously in a 3:3:1:1 ratio (sclerostinbinding agent intravenously: sclerostin binding agent subcutaneously:placebo intravenously: placebo subcutaneously) at a dose level of 1mg/kg or 10 mg/kg (reduced to 5 mg/kg), respectively. For cohorts 3c and6c, four healthy postmenopausal women were randomized to receive thesclerostin binding agent or placebo intravenously in a 3:1 ratio at adose level of 1 mg/kg or 10 mg/kg (reduced to 5 mg/kg), respectively.

The anti-sclerostin therapy was monitored by measuring the levels ofbone resorption markers and bone formation markers prior toadministration, then at least every week for 12 weekspost-administration. P1NP and BSAP levels were monitored following asingle-dose subcutaneous administration of sclerostin binding agent inhealthy, postmenopausal women (see FIGS. 1 and 2). Subjects dosed at 0.1mg/kg and 0.3 mg/kg enjoyed the least elevation of P1NP or BSAP levels(e.g., levels increased less than 20%).

P1NP levels in subjects given 1 mg/kg increased approximately 20% by Day10 and gradually tapered off to baseline around Day 56, while BSAPlevels peaked at Day 14 at about 30% above baseline. P1NP and BSAPlevels in subjects given 3 mg/kg peaked at Day 21 at approximately 100%(P1NP) and 60% (BSAP) increase from baseline, and returned to baselineabout Day 56. In subjects administered 5 mg/kg, the level of P1NP roseto about 140% above baseline at Day 14 post-administration, and remainedelevated at Day 77. In other words, the level of P1NP increased about140% by two weeks post-treatment. BSAP rose to about 115% above baselineand remained elevated at Day 84. Similarly, administration of 10 mg/kgtriggered a 180% increase in P1NP levels at about Day 28. P1NP levelsremained elevated throughout the monitoring period. Subjectsadministered 10 mg/kg demonstrated a peak increase of BSAP levels at Day21 (125% baseline for 3 weeks post-administration), which also remainedelevated at Day 84. The results of the study are illustrated in FIGS. 1and 2.

Osteocalcin also was monitored following a single-dose, subcutaneousadministration of sclerostin binding agent in healthy, postmenopausalwomen (see FIG. 3). Subjects given less than 1 mg/kg experienced littleelevation of Osteocalcin. Osteocalcin levels fluctuated in patientsadministered 1 mg/kg, peaking at about 30% above baseline at Days 21 and35. Osteocalcin levels peaked at about 100% above baseline at Day 21 insubjects administered 3 mg/kg, and levels remained elevated until aboutDay 56. Likewise, administration of 5 mg/kg sclerostin binding agentresulted in a 140% increase in osteocalcin levels at day 28, whichlevels remained at Day 84. Subjects dosed at 10 mg/kg demonstrated apeak osteocalcin level of about 180% above baseline at Day 35.Osteocalcin levels remained elevated above baseline until at least aboutDay 77.

Levels of the bone resorptive marker sCTx also were monitored (see FIG.4). Subjects administered placebo and 0.1 mg/kg demonstrated modestdecreases in sCTx levels (e.g., less than 20%). Administration of 0.3mg/kg of sclerostin binding agent reduced sCTx levels by about 20% byDay 21 (i.e., sCTX levels were reduced about 20% by two weeks aftertreatment). Levels fluctuated in subjects dosed at 1 mg/kg but reachedabout 30% below baseline at Days 10, 28, and 49. Levels in subjectsadministered 3 mg/kg, 5 mg/kg, and 10 mg/kg fell lowest at Day 14 toabout 35%, 55%, and 55% below baseline, respectively, and levelsremained below baseline when monitored thereafter. A comparison of thelevels of all monitored biomarkers is provided in FIG. 5.

Serum ionized calcium levels were monitored following a single,subcutaneous dose of sclerostin binding agent in healthy, postmenopausalwomen (see FIG. 6). Remarkably, ionized calcium levels did not fluctuatedramatically at any dosage. Indeed, all subjects (including thosereceiving placebo) experienced a modest transient decrease in serumionized calcium of approximately 5% during the monitoring period.

Finally, bone mineral density was measured in the spine and hip ofhealthy, postmenopausal women receiving 1 mg/kg, 3 mg/kg, 5 mg/kg, or 10mg/kg sclerostin binding agent (see FIG. 7). Significant increases inBMD were observed in the spine, for example, at Days 28, 56, and 84,particularly in patients receiving 5 mg/kg and 10 mg/kg. BMD in the hipincreased less than that of the spine, but BMD was elevated at Day 56 inpatients administered 3 mg/kg, 5 mg/kg, and 10 mg/kg. BMD was furtherelevated at Day 84 in patients dosed at 5 mg/kg and 10 mg/kg.

This example illustrates the ability of the inventive method to reducelevels of a marker of bone resorption, elevate levels of markers of boneformation, and increase bone mineral density without dramaticalterations in serum calcium. The therapeutic effect of a single dose ofsclerostin binding agent is long-lived, with increased bone formationmarker levels and decreased bone resorptive marker levels continuing tobe observed at 84 days (12 weeks) post treatment. Furthermore, datadescribed herein suggests that the therapeutic efficacy of the inventionhave significant advantages compared to other treatments by “uncoupling”bone formation and bone resorption to maximize bone formation andmineralization in vivo.

All of the references cited herein, including patents, patentapplications, literature publications, and the like, are herebyincorporated in their entireties by reference.

While this invention has been described with an emphasis upon preferredembodiments, it will be obvious to those of ordinary skill in the artthat variations of the preferred compounds and methods may be used andthat it is intended that the invention may be practiced otherwise thanas specifically described herein. Accordingly, this invention includesall modifications encompassed within the spirit and scope of theinvention as defined by the following claims.

1. A method for inhibiting bone resorption in a subject, the methodcomprising administering to the subject a sclerostin binding agent in anamount from about 1 mg/kg to about 10 mg/kg, wherein the amount iseffective to reduce serum level of C-telopeptide of type I collagen(CTX) by at least 20%, compared to pre-treatment or normal levels, by 3weeks after treatment begins, and wherein bone resorption is inhibited.2. The method of claim 1, wherein the amount of sclerostin binding agentincreases a marker of bone formation selected from the group consistingof serum level of bone-specific alkaline phosphatase (BSAP), serum levelof amino-terminal extension of peptide of procollagen type 1 (PINP), andserum level of osteocalcin (OstCa), by at least 20%, compared topre-treatment or normal levels, by 3 weeks after treatment begins. 3.The method of claim 1, wherein the amount of sclerostin binding agentdoes not result in hypocalcemia or hypercalcemia.
 4. The method of claim1, wherein an amount of sclerostin binding agent is administered to thesubject once every two weeks.
 5. The method of claim 1, wherein anamount of sclerostin binding agent is adminstered to the subject once amonth.
 6. The method of claim 1, where the sclerostin binding agentcross-blocks the binding of at least one of antibodies Ab-A, Ab-B, Ab-C,Ab-D, Ab-1, Ab-2, Ab-3, Ab-4, Ab-5, Ab-6, Ab-7, Ab-8, Ab-9, Ab-10,Ab-11, Ab-12, Ab-13, Ab-14, Ab-15, Ab-16, Ab-17, Ab-18, Ab-19, Ab-20,Ab-21, Ab-22, Ab-23, and Ab-24 to sclerostin.
 7. The method of claim 1,wherein the sclerostin binding agent is cross-blocked from binding tosclerostin by at least one of antibodies Ab-A, Ab-B, Ab-C, Ab-D, Ab-1,Ab-2, Ab-3, Ab-4, Ab-5, Ab-6, Ab-7, Ab-8, Ab-9, Ab-10, Ab-11, Ab-12,Ab-13, Ab-14, Ab-15, Ab-16, Ab-17, Ab-18, Ab-19, Ab-20, Ab-21, Ab-22,Ab-23, and Ab-24.
 8. The method of claim 1, wherein the sclerostinbinding agent is an antibody or fragment thereof that demonstrates abinding affinity for sclerostin of SEQ ID NO: 1 of less than or equal to1×10⁻⁷ M.
 9. The method of claim 8, wherein the antibody or fragmentthereof comprises: a) CDR sequences of SEQ ID NOs:54, 55, and 56 and CDRsequences of SEQ ID NOs:51, 52, and 53; b) CDR sequences of SEQ IDNOs:60, 61, and 62 and CDR sequences of SEQ ID NOs:57, 58, and 59; c)CDR sequences of SEQ ID NOs:48, 49, and 50 and CDR sequences of SEQ IDNOs:45, 46, and 47; d) CDR sequences of SEQ ID NOs:42, 43, and 44 andCDR sequences of SEQ ID NOs:39, 40, and 41; e) CDR sequences of SEQ IDNOs:275, 276, and 277 and CDR sequences of SEQ ID NOs:287, 288, and 289;f) CDR sequences of SEQ ID NOs:278, 279, and 280 and CDR sequences ofSEQ ID NOs:290, 291, and 292; g) CDR sequences of SEQ ID NOs:78, 79, and80 and CDR sequences of SEQ ID NOs: 245, 246, and 247; h) CDR sequencesof SEQ ID NOs:81, 99, and 100 and CDR sequences of SEQ ID NOs:248, 249,and 250; i) CDR sequences of SEQ ID NOs:101, 102, and 103 and CDRsequences of SEQ ID NOs:251, 252, and 253; j) CDR sequences of SEQ IDNOs:104, 105, and 106 and CDR sequences of SEQ ID NOs:254, 255, and 256;k) CDR sequences of SEQ ID NOs:107, 108, and 109 and CDR sequences ofSEQ ID NOs:257, 258, and 259; l) CDR sequences of SEQ ID NOs:110, 111,and 112 and CDR sequences of SEQ ID NOs:260, 261, and 262; m) CDRsequences of SEQ ID NOs:281, 282, and 283 and CDR sequences of SEQ IDNOs:293, 294, and 295; n) CDR sequences of SEQ ID NOs:113, 114, and 115and CDR sequences of SEQ ID NOs:263, 264, and 265; o) CDR sequences ofSEQ ID NOs:284, 285, and 286 and CDR sequences of SEQ ID NOs:296, 297,and 298; p) CDR sequences of SEQ ID NOs:116, 237, and 238 and CDRsequences of SEQ ID NOs:266, 267, and 268; q) CDR sequences of SEQ IDNOs:239, 240, and 241 and CDR sequences of SEQ ID NOs:269, 270, and 271;r) CDR sequences of SEQ ID NOs:242, 243, and 244 and CDR sequences ofSEQ ID NOs:272, 273, and 274; or s) CDR sequences of SEQ ID NOs:351,352, and 353 and CDR sequences of SEQ ID NOs:358, 359, and
 360. 10. Themethod of claim 9, wherein the antibody or fragment thereof comprisesCDRH-1, CDR-H2, CDR-H3, CDR-L1 CDR-L2 and CDR-L3 wherein (a) CDR-H1 isSEQ ID NO:245, CDR-H2 is SEQ ID NO:246, CDR-H3 is SEQ ID NO:247, CDR-L1is SEQ ID NO:78, CDR-L2 is SEQ ID NO:79 and CDR-L3 is SEQ ID NO:80; or(b) CDR-H1 is SEQ ID NO:269, CDR-H2 is SEQ ID NO:270, CDR-H3 is SEQ IDNO:271, CDR-L1 is SEQ ID NO:239, CDR-L2 is SEQ ID NO:240 and CDR-L3 isSEQ ID NO:241.
 11. The method of claim 8, wherein the antibody is ahuman antibody, a humanized antibody, a monoclonal antibody, or achimeric antibody.
 12. A method for increasing bone mineral density in asubject, the method comprising administering to the subject a sclerostinbinding agent in an amount from about 1 mg/kg to about 10 mg/kg, whereinthe amount is effective to (a) reduce serum level of CTX by at least 20%compared to pre-treatment or normal levels, by 3 weeks after treatmentbegins, and (b) increase serum level of a bone formation marker selectedfrom the group consisting of serum level of bone-specific alkalinephosphatase (BSAP), serum level of amino-terminal extension of peptideof procollagen type 1 (PINP), and serum level of osteocalcin (OstCa), byat least 20%, compared to pre-treatment or normal levels, by 3 weeksafter treatment begins, and wherein bone mineral density is increased.13. The method of claim 12, wherein hip, spine, wrist, finger, shin boneand/or heel bone mineral density is increased by at least about 1%. 14.The method of claim 13, wherein bone mineral density in the spine isincreased by at least about 1%.
 15. The method of claim 12, wherein bonemineral density is increased to the range of about 1 to 2.5 standarddeviations below the normal bone mineral density of a healthy youngadult.
 16. The method of claim 12, wherein bone mineral density isincreased to the range of about 0 to 1 standard deviations below thenormal bone mineral density of a healthy young adult.
 17. The method ofclaim 12, wherein an amount of sclerostin binding agent is administeredto the subject once every two weeks.
 18. The method of claim 12, whereinan amount of sclerostin binding agent is adminstered to the subject oncea month.
 19. The method of claim 12, where the sclerostin binding agentcross-blocks the binding of at least one of antibodies Ab-A, Ab-B, Ab-C,Ab-D, Ab-1, Ab-2, Ab-3, Ab-4, Ab-5, Ab-6, Ab-7, Ab-8, Ab-9, Ab-10,Ab-11, Ab-12, Ab-13, Ab-14, Ab-15, Ab-16, Ab-17, Ab-18, Ab-19, Ab-20,Ab-21, Ab-22, Ab-23, and Ab-24 to sclerostin.
 20. The method of claim12, wherein the sclerostin binding agent is cross-blocked from bindingto sclerostin by at least one of antibodies Ab-A, Ab-B, Ab-C, Ab-D,Ab-1, Ab-2, Ab-3, Ab-4, Ab-5, Ab-6, Ab-7, Ab-8, Ab-9, Ab-10, Ab-11,Ab-12, Ab-13, Ab-14, Ab-15, Ab-16, Ab-17, Ab-18, Ab-19, Ab-20, Ab-21,Ab-22, Ab-23, and Ab-24.
 21. The method of claim 12, wherein thesclerostin binding agent is an antibody or fragment thereof thatdemonstrates a binding affinity for sclerostin of SEQ ID NO: 1 of lessthan or equal to 1×10⁻⁷ M.
 22. The method of claim 21, wherein theantibody or fragment thereof comprises: a) CDR sequences of SEQ IDNOs:54, 55, and 56 and CDR sequences of SEQ ID NOs:51, 52, and 53; b)CDR sequences of SEQ ID NOs:60, 61, and 62 and CDR sequences of SEQ IDNOs:57, 58, and 59; c) CDR sequences of SEQ ID NOs:48, 49, and 50 andCDR sequences of SEQ ID NOs:45, 46, and 47; d) CDR sequences of SEQ IDNOs:42, 43, and 44 and CDR sequences of SEQ ID NOs:39, 40, and 41; e)CDR sequences of SEQ ID NOs:275, 276, and 277 and CDR sequences of SEQID NOs:287, 288, and 289; f) CDR sequences of SEQ ID NOs:278, 279, and280 and CDR sequences of SEQ ID NOs:290, 291, and 292; g) CDR sequencesof SEQ ID NOs:78, 79, and 80 and CDR sequences of SEQ ID NOs: 245, 246,and 247; h) CDR sequences of SEQ ID NOs:81, 99, and 100 and CDRsequences of SEQ ID NOs:248, 249, and 250; i) CDR sequences of SEQ IDNOs:101, 102, and 103 and CDR sequences of SEQ ID NOs:251, 252, and 253;j) CDR sequences of SEQ ID NOs:104, 105, and 106 and CDR sequences ofSEQ ID NOs:254, 255, and 256; k) CDR sequences of SEQ ID NOs:107, 108,and 109 and CDR sequences of SEQ ID NOs:257, 258, and 259; l) CDRsequences of SEQ ID NOs:110, 111, and 112 and CDR sequences of SEQ IDNOs:260, 261, and 262; m) CDR sequences of SEQ ID NOs:281, 282, and 283and CDR sequences of SEQ ID NOs:293, 294, and 295; n) CDR sequences ofSEQ ID NOs:113, 114, and 115 and CDR sequences of SEQ ID NOs:263, 264,and 265; o) CDR sequences of SEQ ID NOs:284, 285, and 286 and CDRsequences of SEQ ID NOs:296, 297, and 298; p) CDR sequences of SEQ IDNOs:116, 237, and 238 and CDR sequences of SEQ ID NOs:266, 267, and 268;q) CDR sequences of SEQ ID NOs:239, 240, and 241 and CDR sequences ofSEQ ID NOs:269, 270, and 271; r) CDR sequences of SEQ ID NOs:242, 243,and 244 and CDR sequences of SEQ ID NOs:272, 273, and 274; or s) CDRsequences of SEQ ID NOs:351, 352, and 353 and CDR sequences of SEQ IDNOs:358, 359, and
 360. 23. The method of claim 22, wherein the antibodyor fragment thereof comprises CDRH-1, CDR-H2, CDR-H3, CDR-L1 CDR-L2 andCDR-L3 wherein (a) CDR-H1 is SEQ ID NO:245, CDR-H2 is SEQ ID NO:246,CDR-H3 is SEQ ID NO:247, CDR-L1 is SEQ ID NO:78, CDR-L2 is SEQ ID NO:79and CDR-L3 is SEQ ID NO:80; or (b) CDR-H1 is SEQ ID NO:269, CDR-H2 isSEQ ID NO:270, CDR-H3 is SEQ ID NO:271, CDR-L1 is SEQ ID NO:239, CDR-L2is SEQ ID NO:240 and CDR-L3 is SEQ ID NO:241.
 24. The method of claim21, wherein the antibody is a human antibody, a humanized antibody, amonoclonal antibody, or a chimeric antibody.
 25. A method for treating abone-related disorder in a subject, the method comprising (a)administering to the subject a sclerostin binding agent in an amountfrom about 1 mg/kg to about 10 mg/kg for a first period of time, whereinthe amount is effective to increase bone mineral density at the hip,spine, wrist, finger, shin bone and/or heel by at least about 3%, and(b) administering to the subject a sclerostin binding agent in an amountof from about 1 mg/kg to about 10 mg/kg for a second period of timeeffective to maintain bone mineral density.
 26. The method of claim 25,wherein the first period of time is 3 months or less.
 27. The method ofclaim 25, wherein the second period of time is at least 6 months. 28.The method of claim 25, wherein bone mineral density in the spine isincreased by at least about 3%.
 29. The method of claim 25, wherein anamount of sclerostin binding agent is administered to the subject onceevery two weeks.
 30. The method of claim 25, wherein an amount ofsclerostin binding agent is adminstered to the subject once a month. 31.The method of claim 25 in which the bone-related disorder is selectedfrom the group consisting of achondroplasia, cleidocranial dysostosis,enchondromatosis, fibrous dysplasia, Gaucher's Disease, hypophosphatemicrickets, Marfan's syndrome, multiple hereditary exotoses,neurofibromatosis, osteogenesis imperfecta, osteopetrosis,osteopoikilosis, sclerotic lesions, pseudoarthrosis, pyogenicosteomyelitis, periodontal disease, anti-epileptic drug induced boneloss, primary and secondary hyperparathyroidism, familialhyperparathyroidism syndromes, weightlessness induced bone loss,osteoporosis in men, postmenopausal bone loss, osteoarthritis, renalosteodystrophy, infiltrative disorders of bone, oral bone loss,osteonecrosis of the jaw, juvenile Paget's disease, melorheostosis,metabolic bone diseases, mastocytosis, sickle cell anemia/disease, organtransplant related bone loss, kidney transplant related bone loss,systemic lupus erythematosus, ankylosing spondylitis, epilepsy, juvenilearthritides, thalassemia, mucopolysaccharidoses, Fabry Disease, TurnerSyndrome, Down Syndrome, Klinefelter Syndrome, leprosy, Perthes'Disease, adolescent idiopathic scoliosis, infantile onset multi-systeminflammatory disease, Winchester Syndrome, Menkes Disease, Wilson'sDisease, ischemic bone disease (such as Legg-Calve-Perthes disease,regional migratory osteoporosis), anemic states, conditions caused bysteroids, glucocorticoid-induced bone loss, heparin-induced bone loss,bone marrow disorders, scurvy, malnutrition, calcium deficiency,osteoporosis, osteopenia, alcoholism, chronic liver disease,postmenopausal state, chronic inflammatory conditions, rheumatoidarthritis, inflammatory bowel disease, ulcerative colitis, inflammatorycolitis, Crohn's disease, oligomenorrhea, amenorrhea, pregnancy,diabetes mellitus, hyperthyroidism, thyroid disorders, parathyroiddisorders, Cushing's disease, acromegaly, hypogonadism, immobilizationor disuse, reflex sympathetic dystrophy syndrome, regional osteoporosis,osteomalacia, bone loss associated with joint replacement, HIVassociated bone loss, bone loss associated with loss of growth hormone,bone loss associated with cystic fibrosis, chemotherapy associated boneloss, tumor induced bone loss, cancer-related bone loss, hormoneablative bone loss, multiple myeloma, drug-induced bone loss, anorexianervosa, disease associated facial bone loss, disease associated cranialbone loss, disease associated bone loss of the jaw, disease associatedbone loss of the skull, bone loss associated with aging, facial boneloss associated with aging, cranial bone loss associated with aging, jawbone loss associated with aging, skull bone loss associated with aging,and bone loss associated with space travel.
 32. The method of claim 25,where the sclerostin binding agent cross-blocks the binding of at leastone of antibodies Ab-A, Ab-B, Ab-C, Ab-D, Ab-1, Ab-2, Ab-3, Ab-4, Ab-5,Ab-6, Ab-7, Ab-8, Ab-9, Ab-10, Ab-11, Ab-12, Ab-13, Ab-14, Ab-15, Ab-16,Ab-17, Ab-18, Ab-19, Ab-20, Ab-21, Ab-22, Ab-23, and Ab-24 tosclerostin.
 33. The method of claim 25, wherein the sclerostin bindingagent is cross-blocked from binding to sclerostin by at least one ofantibodies Ab-A, Ab-B, Ab-C, Ab-D, Ab-1, Ab-2, Ab-3, Ab-4, Ab-5, Ab-6,Ab-7, Ab-8, Ab-9, Ab-10, Ab-11, Ab-12, Ab-13, Ab-14, Ab-15, Ab-16,Ab-17, Ab-18, Ab-19, Ab-20, Ab-21, Ab-22, Ab-23, and Ab-24.
 34. Themethod of claim 25, wherein the sclerostin binding agent is an antibodyor fragment thereof that demonstrates a binding affinity for sclerostinof SEQ ID NO: 1 of less than or equal to 1×10⁻⁷ M.
 35. The method ofclaim 34, wherein the antibody or fragment thereof comprises: a) CDRsequences of SEQ ID NOs:54, 55, and 56 and CDR sequences of SEQ IDNOs:51, 52, and 53; b) CDR sequences of SEQ ID NOs:60, 61, and 62 andCDR sequences of SEQ ID NOs:57, 58, and 59; c) CDR sequences of SEQ IDNOs:48, 49, and 50 and CDR sequences of SEQ ID NOs:45, 46, and 47; d)CDR sequences of SEQ ID NOs:42, 43, and 44 and CDR sequences of SEQ IDNOs:39, 40, and 41; e) CDR sequences of SEQ ID NOs:275, 276, and 277 andCDR sequences of SEQ ID NOs:287, 288, and 289; f) CDR sequences of SEQID NOs:278, 279, and 280 and CDR sequences of SEQ ID NOs:290, 291, and292; g) CDR sequences of SEQ ID NOs:78, 79, and 80 and CDR sequences ofSEQ ID NOs: 245, 246, and 247; h) CDR sequences of SEQ ID NOs:81, 99,and 100 and CDR sequences of SEQ ID NOs:248, 249, and 250; i) CDRsequences of SEQ ID NOs:101, 102, and 103 and CDR sequences of SEQ IDNOs:251, 252, and 253; j) CDR sequences of SEQ ID NOs:104, 105, and 106and CDR sequences of SEQ ID NOs:254, 255, and 256; k) CDR sequences ofSEQ ID NOs:107, 108, and 109 and CDR sequences of SEQ ID NOs:257, 258,and 259; l) CDR sequences of SEQ ID NOs:110, 111, and 112 and CDRsequences of SEQ ID NOs:260, 261, and 262; m) CDR sequences of SEQ IDNOs:281, 282, and 283 and CDR sequences of SEQ ID NOs:293, 294, and 295;n) CDR sequences of SEQ ID NOs:113, 114, and 115 and CDR sequences ofSEQ ID NOs:263, 264, and 265; o) CDR sequences of SEQ ID NOs:284, 285,and 286 and CDR sequences of SEQ ID NOs:296, 297, and 298; p) CDRsequences of SEQ ID NOs:116, 237, and 238 and CDR sequences of SEQ IDNOs:266, 267, and 268; q) CDR sequences of SEQ ID NOs:239, 240, and 241and CDR sequences of SEQ ID NOs:269, 270, and 271; r) CDR sequences ofSEQ ID NOs:242, 243, and 244 and CDR sequences of SEQ ID NOs:272, 273,and 274; or s) CDR sequences of SEQ ID NOs:351, 352, and 353 and CDRsequences of SEQ ID NOs:358, 359, and
 360. 36. The method of claim 35,wherein the antibody or fragment thereof comprises CDRH-1, CDR-H2,CDR-H3, CDR-L1 CDR-L2 and CDR-L3 wherein (a) CDR-H1 is SEQ ID NO:245,CDR-H2 is SEQ ID NO:246, CDR-H3 is SEQ ID NO:247, CDR-L1 is SEQ IDNO:78, CDR-L2 is SEQ ID NO:79 and CDR-L3 is SEQ ID NO:80; or (b) CDR-H1is SEQ ID NO:269, CDR-H2 is SEQ ID NO:270, CDR-H3 is SEQ ID NO:271,CDR-L1 is SEQ ID NO:239, CDR-L2 is SEQ ID NO:240 and CDR-L3 is SEQ IDNO:241.
 37. The method of claim 34, wherein the antibody is a humanantibody, a humanized antibody, a monoclonal antibody, or a chimericantibody.
 38. A method of treating a bone-related disorder in a humansuffering from or at risk of hypocalcemia or hypercalcemia, the methodcomprising administering to the human a sclerostin binding agent in atherapeutically effective amount from about 1 mg/kg to about 10 mg/kg.39. The method of claim 38, wherein the hypocalcemia or hypercalcemiaresults from chronic kidney disease, renal failure, primary or secondaryhyperparathyroidism, pseudohyperparathyroidism, hypoparathyroidism,pseudohypoparathyroidism, magnesium depletion, severe hypermagnesemia,vitamin D deficiency, hyperphosphatemia, acute pancreatitis, hungry bonesyndrome, chelation, osteoblastic metastases, sepsis, surgery,chemotherapy, neoplasia syndrome, hypoparathyroidism, familialhypocalciuric hypercalcemia, sarcoidosis, tuberculosis, berylliosis,histoplasmosis, Candidiasis, Coccidioidomycosis, histiocytosis X,Hodgkin's or Non-Hodgkin's lymphoma, Crohn's disease, Wegener'sgranulomatosis, pneumonia, silicone-induced granulomas, administrationof thiazide diuretics or lithium, or immobilization.
 40. A method oftreating a bone-related disorder in (a) a human in which treatment witha parathyroid hormone or analog thereof is contraindicated or (b) ahuman in which treatment with bisphosphonate is contraindicated, themethod comprising administering to the human a therapeutically effectiveamount of a sclerostin binding agent from about 1 mg/kg to about 10mg/kg.